staining procedure was in 1883, in a treatise by Carl Friedlander on the micrococci

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1 THE GRAM STAIN JAMES W. BARTHOLOMEW AND TOD MITTWER Department of Bacteriology, University of Southern California, Los Angeles 7, California CONTENTS I. INTRODUCTION... 1 II. THE GRAM PROCEDURE The primary stain The mordant The decolorizer The counterstain Other factors... 6 III. CORRELATION OF GRAM REACTION WITH PHYSIOLOGICAL CHARACTERISTICS... 8 IV. DEGREE OF GRAM POSITIVITY Studies of decolorization time. Gram Dauer character Conversion of gram positive cells to gram negative Addition of gram positivity to cells previously rendered gram negative Conversion of cells normally gram negative to the gram positive state V. THE MECHANISM OF GRAM DIFFERENTIATION Chemical theories of gram positivity The isoelectric point concept Theories involving permeability VI. THE SITE OF THE GRAM REACTION.20 VII. CONCLUSIONS VIII. REFERENCES I. INTRODUCTION During the early days of bacteriology the detection of bacterial cells in tissues was difficult since most of the staining methods used colored the tissue and the bacterial cells equally. Christian Gram (as a co-worker of Dr. Friedlander in the municipal hospital of Berlin) attempted the development of a procedure which would differentially stain schizomycetes from tissue cells. Gram began his work with pneumococci in the lungs of human pneumonia victims and with lung tissue from experimental animals. The first published mention of the now famous gram staining procedure was in 1883, in a treatise by Carl Friedlander on the micrococci of pneumonia (59). This paper contained a brief description of Gram's staining procedure. The following year Gram published his staining method in detail (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 attempted to obtain blue nuclei and brown cytoplasm in kidney sections by staining first with gentian violet and then with iodine-potassium iodide solution. This application of iodine, instead of the intended result, rendered destaining and clearing of the gentian violet, ordinarily very difficult, quite easy by the subsequent alcohol step. In Gram's words, "The experiments resulted from the accidental observation that aniline-gentian violet preparations of tissues, after treatment with iodine-potassium iodide, are completely and rapidly decolorized in al-

2 2 JAMES W. BARTHOLOMEW AND TOD MITTWER [VOL. 16 cohol" (63). During this work Gram noticed the outstanding resistance of some bacterial cells to decolorization. The now famous, extremely useful, and enigmatic gram staining procedure was a natural development from these observations. 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 lung tissue he used Ehrlich's aniline-gentian violet, Lugol's iodine, absolute alcohol for decolorization, and Bismarck brown as a counterstain. Gram used his procedure primarily to stain bacteria in tissues, but he also demonstrated its applicability to smears of pure cultures. The counterstain was used to color the tissue cells, including their nuclei, differentially from the bacteria. Although Gram observed that certain types of bacterial cells such as typhoid bacilli were decolorized by his procedure and took the color of the counterstain, he did not divide 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 to uncertainty resulting from his observation that while most of his pneumococci retained the gentian violet, some strains of pneumonia-producing bacteria were decolorized. Probably the first diagnostic use to which Gram's stain was put was in 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 valuable step in diagnosis and classification. The ability to retain the primary dye in the gram stain is a unique characteristic restricted to but a small fraction of the materials of nature. Most plant and animal cells stain gram negatively. The gram 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 the poliedes of the silkworm, have been reported as weakly to strongly gram positive (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 procedure involved can often be questioned. In addition, the reaction to the gram procedure has been shown to correlate with important chemical and physiological characteristics of the cell (see Section 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 out of print and difficult to obtain. In other papers where this stain has been considered (36, 47, 77, 100, 118, 147) the review treatment of the subject is brief and incomplete. In the present review the authors will condense and bring up to 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 aniline gentian violet, an aqueous solution of iodine-potassium iodide, absolute alcohol as a decolorizer, and sometimes Bismarck brown as a counterstain. The method is now fundamentally the same; however, a long series of important modifica-

3 1952] THE GRAM STAIN 3 tions has resulted in procedures which produce more reliable results, and which are much more convenient than the original. The best comparisons of the various proposed modifications have been conducted by Hucker and Conn (77). These meticulous and extensive studies have given workers some scientific basis for the choice of a gram stain method. Of the modifications suggested many gave good, 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 reproducible substance than the old dye mixtures called gentian violet. The primary dye is a stable solution, and it may contain a mordant such as ammonium oxalate for constancy of action; or sodium bicarbonate may be added just before use to intensify the uptake of the color. The staining step and the iodine step may be carried out at an alkaline ph by addition of sodium bicarbonate or sodium hydroxide to the dye, the iodine solution, or to both. This avoids poor results which are sometimes due to the acid state of the organisms, their suspension medium (e.g., pus), or the reagents (24, 89, 124). Alcohol (95%) is most commonly used as a decolorizer, although acetone and acetone-alcohol mixtures are sometimes used and are excellent (89, 100). The dilution of the alcohol with water, by repeated use or by water remaining from a preceding wash step, is to be avoided. Safranin is the most popular of the possible counterstains. These features will be discussed in more detail in the following paragraphs. There is no gram procedure which can be referred to as the best for all laboratories and for all situations. Hucker and Conn's recommendation (77) that the worker adopt at least two of the well-accepted methods, practice them until he is familiar with their characteristics, and use controls of organisms with known gram reactions is excellent counsel. This plan will give much better results than constant 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 of stability of the Ehrlich's aniline gentian violet solution. This instability was due to a slowly formed precipitate which results from the interaction of the dye and aniline. Among the attempts to increase this stability have been the suggestions of various nonprecipitate forming substitutes for aniline such as the slow addition of phenol (41, 95, 96, 102, 110), boric acid (88), aniline sulfate or aniline oxalate (2), glycerol (78), oxalic acid (109), or even the complete omission of the mordant from the primary stain (24, 89, 90). The most widely used formulas in the United States are those of Hucker (75) and Kopeloff-Beerman (89). Hucker's crystal violet contains ammonium oxalate and is very stable. In addition, Hucker and Conn demonstrated that ammonium oxalate in the formula gave 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 crystal violet but added sodium bicarbonate to small quantities of stock solutions just before use, or even on the slide itself. The resulting alkaline ph for staining is known to contribute to the clarity of the differentiation.

4 4 JAMES W. BARTHOLOMEW AND TOD MITTWER [VOL. 16 The specificity of crystal or gentian violet as the primary stain has long been a matter of dispute. The problem has recently been studied by Bartholomew and Mittwer (9, 10), who found that a large number of basic dyes, especially of 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 to crystal 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 dyes with weak tinctorial properties, were found to be useless. 2. The mordant. In this section the term mordant is used to describe iodinepotassium iodide solution or a substitute for this solution as used in the gram procedure. It is to be distinguished from substances added to the primary dye which are also called mordants. Gram's choice of iodine, which he expected to form a background color in his sections, was fortunate. No other reagent can be substituted for the best and most reliable results (106). However, iodine is not absolutely specific, and substitutes for it are possible. Suggested substitutes for the iodine solution have included bromine, picric acid, any metallic iodide such as that of Mg, Na, As, Al, Fe, Hg, or Zn, ammonium picrate, salts such as sodium chloride, 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 these and others have recently been retested under standardized conditions (106). Since most give poor results or irregular results with the exception of picric acid and mercuric iodide or chloride, none of these suggested substitutes can be recommended. Many formulas for the iodine solution contain an alkalizing agent such as sodium bicarbonate (124) or sodium hydroxide (89) because Sheppe and Constable (124) demonstrated that on storage the iodine solution develops sufficient acidity through oxidation to cause occasional errors in gram differentiation. In view of this work and the well-known influence of ph on a satisfactory gram stain (25), the use of an alkaline iodine solution can be recommended. That this is not often a serious cause of error has been demonstrated, however, by the present reviewers, who acidified the iodine solution to ph's of less than 1 and observed a slight increase in the percentage of the gram negative cells normally found 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 sufficiently controlled (14, 15, 17, 73, 91, 106). Although significant for research on the theory, it would be practically impossible to determine the gram reaction of an unknown organism in this manner. 3. The decolorizer. The application of the decolorizer is the most critical stage of the gram procedure. There is danger from both over and under decolorization; therefore, the decolorizer and the technique should be as carefully standardized as possible. Ethyl alcohol has been one of the most popular decolorizers. According to Neide (108) addition of water to the alcohol increases its rate of decolorization up to 40% alcohol; he considered 60% alcohol to be too rapid a

5 1952] THE GRAM STAIN 5 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 little practical difference between the decolorization effect of 95% and absolute alcohol. These, however, gave more dependable results than alcohol solutions contalning more water. Thus, it is necessary to conduct the procedure in such a way that the alcohol does not change its dilution. Blotting dry before decolorization is recommended (128) and is probably desirable (45). The results reported by Hucker and Conn (77) indicate that only 95% or absolute alcohol should be used when alcohol is applied as the decolorizer in the gram stain. Other alcohols have been studied, and it has been reported that the more complex the alcohol, the slower the decolorization action. As the carbon chain lengthens, decolorization is slower. Kisskalt (84) found decolorization power decreasing in the following order: methyl, ethyl, propyl, butyl, and amyl alcohol. Conn (40) found a mixture of equal parts of methyl and isopropyl alcohols to have very similar decolorization properties to ethyl alcohol. In practice, however, no known advantage can be gained by substituting the higher alcohols for ethyl 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 must be used with some care, but the validity of its use in the gram stain has been amply demonstrated by Lillie (100) and others. Other decolorizers suggested have included aniline (146) which was reported to give more dependable results than ethyl alcohol because it was slower in action and thus more easily controlled; chloroform (129), any surface tension depressant (15), dilute acids, acid alcohols (27, 63), sodium thiosulfate (101), and salt solutions (41). In the hands of the reviewers, only alcohol, acetone, or mixtures of these two have given consistently good results. These latter decolorizers are almost universally used, and the choice of one or the other is of less importance than 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 determinations of the temperature effect are not now available, but it is established that an increase in temperature results in more rapid decolorization (45). The evidence so far presented indicates that controlled decolorization temperature would be of benefit in obtaining consistent results in critical research but probably not necessary under ordinary laboratory conditions. 4. The counterstain. Some investigators of the gram stain have omitted the counterstain in their work (132, 134). It is indeed possible to differentiate some bacteria entirely on the basis of the time necessary for complete or partial decolorization (45, 98, 102, 108). However, this may or may not be related to the true gram differentiation (9, 10, 26, 27, 77, 106). The ability of one dye to replace another has been repeatedly demonstrated for years (12). This power of replacement could be a means by which the counterstain might assume an active role in the gram differentiation (77). Indeed, it is the opinion of some

6 6 JAMES W. BARTHOLOMEW AND TOD MITTWER [VOL. 16 authors (9, 10, 26, 27, 106) that for this reason the.application of the counterstain is a fundamental and necessary step in true gram differentiation. If this is so, then the factors influencing dye replacement assume importance in the gram procedure. Since the replacement tendency is in proportion to the concentration of the second dye and time of application (12), these two factors should be controlled. The influence of the concentration of the counterstain on gram differentiation was shown by Lasseur and Schmitt (98) who reported that 0.031% and 0.5% solutions of basic fuchsin gave greatly different results. The 0.5% solution resulted in many gram positive cells appearing red since it had sufficient replacement power to replace the primary dye remaining after decolorization. Also, if no counterstain is used, gram negative cells may appear gram positive (9, 10, 106). Thus, the counterstain is a definite part of the gram procedure. It is usually a weak solution of about 0.25% dye; its time of application 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 as Bismarck 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 "are so powerful in their action that they tend to decolorize some of the gram positive organisms." Their experiments on the reliability of various counterstains indicated that "pyronin and Bismarck brown are the best counterstains, while eosin and safranin are fair substitutes." As good contrast is also an important factor in practical work, this consideration led them to a personal preference for safranin, a choice confirmed by the experiences of most workers. 5. Other factors. Although many authors have observed that the culture medium 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 exact statement to be made. Most of the recorded variations have been observed on rather unusual media, such as the reported conversion of Bacillus subtilis to a gram negative state by growth on cerium agar (126), the reported conversion of Escherichia coli to the gram positive state by growth in a liquid medium containing a maximum amount of glucose, MgSO4, or NaCl (14, 15). On the other hand, Lasseur and Schmitt (98) found it impossible to change the gram reaction of Micrococcus pyogenes var. aureus by changing its growth medium. It has been reported that starvation of Bacillus cereus, i.e., placing the cells in distilled water at 37 C for 48 hours, results in an increase in the number of gram negative cells in a culture (135). Whether this is due to autolysis, leaching of materials from within 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 of benzimidazole added to Dubos' medium gradually rendered an avian strain of Mycobacterium tuberculosis gram negative as well as non-acid-fast. It is said that

7 1952] THE GRAM STAIN 7 benzimidazole inhibits the synthesis of ribonucleic acid. Experiments of this type should in the future shed more light on the role of the medium in relation to the gram reaction. At present, though, little work is available to indicate whether the influence of abnormal media would be greater or less than the influence of normal variations in the technique used in the gram stain. It appears for the most part that the individual worker would not encounter serious error if he used any medium well accepted for the culture of the species of organism at hand. However, in case of doubt as to the results obtained, two or three media should be used. More study of the influence of growth environment on the gram reaction 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 at temperatures very near the maximum for these organisms. There is no evidence that incubation temperatures which vary only a few degrees from the optimum for growth of the organism could seriously influence the results of the gram procedure. However, since the influence of temperature is not well determined, it is undoubtedly wise to use incubation temperatures near or at the optimum for the organism concerned. All investigators agree that the age of the culture influences the degree of gram positivity of the culture. According to Lasseur and Schmitt (98) there is a stage, which differs for each species of organism, in which the gram positive characteristic is most marked. The differences between young cultures of 24 hours and old cultures of several days are easily demonstrated, and the old cultures are nearly always less gram positive (45, 90, 108). The difference between a culture of one or five hours, or twenty-four and forty-eight hours, however, is much less impressive. Lasseur and Schmitt (98) and Hucker and Conn (77) state that adult cells of approximately 48 hours of age are sometimes more gram positive 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 showed that even at the stage of first sporulation these cells remained gram positive after decolorization for 10 minutes in 95% alcohol. The evidence shows that the custom of using only an 18 to 24 hour culture for the determination of gram characteristics is unwise. Although serious errors due to use of a 12 or 48 hour culture probably would occur only in special instances, it would obviously be better to use at least three different ages of an unknown culture to determine its gram character as urged by Hucker and Conn on the basis of extensive studies of this question (77). The thermophilic organisms must be given special consideration. DeBord (44) showed that spore-forming thermophiles were gram negative by 24 hours and were most gram positive at 8 hours, an understandable result in the light of the greatly accelerated metabolism of the thermophilic species. Six to eight hour cultures, as well as the usual 18 to 24 hour cultures, therefore, should be used in determining the gram reaction of thermophilic species. The degree of gram positivity of cells can be influenced considerably by the

8 8 JAMES W. BARTHOLOMEW AND TOD MITTWER [VOL. 16 method of fixation. Del6tang (45) clearly showed the great differences of decolorization time between yeast cells fixed with alcohol-ether, Bouin solution (picric acid, acetic acid, formalin), acetic sublimate, Miller-formol (potassium dichromate, sodium sulfate, neutralized formalin), and Champy's solution (osmic acid, chromic acid, potassium dichromate). The differences are large, ranging from decolorization times of 10 hours to 40 hours. Del6tang's thorough work confirmed a number of earlier reports (34). Since these fixatives are rarely used in the ordinary gram procedure, they have little interest for those who use heatfixed smears on glass slides. However, the work commands the attention of technicians working with tissue sections. The common practice of using heat to fix slides for gram staining is supported by the work of Nikitin (111), who showed that large variations in the heat fixing step did not influence the results obtained. A note of caution is created by the work of Neide (108), who demonstrated that prolonged heat fixation caused gram positive cells to stain gram negatively, and by the variations in degree of gram positivity found by Del& tang (45) after various degrees of heat fixation. However, small differences in the 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) showed that thick smears of Vibrio cholerae took 40 minutes to be decolorized completely with acetic acid while thin smears took only 13 minutes. For critical research, standard amounts of standard suspensions of organisms should be spread over standard areas of slides in order that individual cells be separated from each other insofar as this is possible (9, 77). In all preparations large masses of grouped organisms should be avoided. Before staining, the slide should be protected against contact with other objects. Any contact with another slide or hard object will create large or small areas of gram negative cells in preparations which would otherwise be gram positive. 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 gram reaction of bacteria with their physiological characteristics. Since then many new correlations have been demonstrated. Table 1 presents most of those known at the present time. The table is necessarily incomplete both in subject content and references. Any attempt to include all the material would prove too bulky and would result in an unwieldy table containing much unreliable and conflicting data. In perusing the material in table 1 it must be kept in mind that almost all of the differences shown are of degree rather than absolute. This is true even for the differential nature of the gram staining procedure itself. It is impossible to compile a table which will give characteristics applicable to all organisms. For instance, the organisms in the genus Neisseria are usually classified as gram negative, but their physiological reactions are more like those of the gram posi-

9 1952] THE GRAM STAIN 9 tive group (7, 127). Thus, table 1 applies to organisms in general but does not necessarily 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) claim such 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, chloroform, 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 correlation does not exist, and that the results of Lasseur might be explained on some other basis, such as a chance choice of bacterial species. In any event, if a correlation between gram character and electrophoretic velocity does exist, it is sufficiently small so as to be missed by several investigators. A similarity in mechanisms of the gram stain and the acid-fast stain has long

10 10 JAMES W. BARTHOLOMEW AND TOD MITTWER [VOL. 16 been suspected (16, 17). This suspicion is based on the many similarities between the 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). Both procedures require a mordant; iodine for the gram stain and phenol for the acidfast 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 is often accompanied by conversion from the gram positive to the gram negative state (6, 92). Media and conditions of growth influence both reactions (87). Since all of these similarities exist, possibly the mechanism of these two differential 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 narrowly separated. If the gram negative and gram positive groups as determined by staining are imposed on a scale representing physiological differences, then overlapping between gram positive and gram negative species would often occur. Churchman and Siegel (37) presented such overlapping for dye sensitivity. Smith (127) demonstrated a similar overlapping for sensitivity to electrolytes and digestion by trypsin or alkali. The best acknowledgment of this gradation between gram positive and gram negative characteristics is the common observation that a reliable gram stain is more the result of a technique than of a step by step procedure. The causes of gram variability can be grouped under two headings: (a) variability due to the staining technique used, and (b) variability inherent in the organisms themselves. The variability attributable to the technique has been considered in an earlier section. It has been shown, however, that even careful standardization of the technique will not prevent conflicting results (77). Some organisms such as yeasts are so gram positive that few errors occur; others such as the diphtheria organism, various aerobic spore-formers, or the gonococcus, often give variable results (72). The gram positive character varies from strong to weak, not only among species but also among different cultures of the same species grown under different conditions (39). Thus, often the true gram reaction of an organism can be obtained only after a considerable period of observation involving 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 introduced to describe those organisms which were not consistent in their reaction to 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 the old gram positive or gram negative groupings. 1. Studies of decolorization time. Gram Dauer character. Neide (108), clearly recognizing the weakness of a strict gram positive or gram negative terminol-

11 1952] THE GRAM STAIN 11 ogy, tried to establish a more definite criterion which he called the Gram Dauer characteristic of an organism, defined as the time necessary to decolorize the cells under standardized conditions after the application of the primary dye and the iodine. Thus, no cell is absolutely gram positive, i.e., it will be decolorized if the alcohol is allowed to act long enough. Neide found that the decolorization time was a constant for each species or strain under standard conditions, and that the decolorization time was sufficiently different between species to make this a valuable differential characteristic. Lasseur and Schmitt (98) and Del6- tang (45) extensively studied the Gram Dauer characteristic and proposed modified techniques to increase the reproducibility of results. In the final analysis the Gram Dauer character demonstrates a gradation of the decolorization times of different species of bacteria. It does not result in grouping of the organisms but shows that all bacteria lie on a broad scale with reference 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 the usual 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 of cells decolorized vs. time results in a rather gently-sloping S-curve (45). Variations in concentration, temperature, and time of application of the reagents, as well as the cultural conditions of the organism, strain used, and the like, all influence the results. Del6tang's method (45) is reproducible under carefully standardized conditions. It has provided valuable data and has demonstrated the variation in degree of gram positivity as a function of species, culture medium, fixatives used, and other factors. It is unfortunate that the procedure is much too painstaking and laborious a process to be used routinely since this quantitative measure of gram positivity would be extremely useful in classification and in research of many kinds. If a simple method of quantitatively determining gram positivity could be found, a real service would be rendered to bacteriology. Another confusing variability in the gram procedure is the presence of gram positive granules in cells otherwise gram negative. An interesting example of this is Thiobacillus thiooxidans (86). It is well known that an overdecolorization of 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 possible that an explanation may lie in the recent findings of Mudd and Smith (107) that bacteria possess "vesicular nuclei containing chromatin." Also Knaysi and his 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 to remain gram positive after the cells themselves had been rendered gram negative. However, very little work has been done on the nature of gram positive granules in general. It may well be that their gram positive property rests on a mechanism involving factors in addition to those which determine the gram characteristic of the cell as a whole (10). The confusion occasioned by these spots is reduced if one determines the gram character of an organism only from

12 12 JAMES W. BARTHOLOMEW AND TOD MITTWER [VOL. 16 the appearance of the cells as a whole, and only after an extensive study of the gram character of the organism involved. 2. Conversion of gram positive cells to gram negative. Almost as soon as the gram positive state was recognized, attempts were made to determine its cause by the use of methods of pretreatment which convert normally gram positive cells to a gram negative state. These attempts have been numerous and often successful. However, they leave behind them a bewildering array of methods used and conflicting results and interpretations. The influence of media and temperature of incubation have been mentioned in an earlier part of this review and need not be 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, for times of from two hours to several days (1, 36, 79, 135, 139). The success of this method has been limited to certain species and culture strains, and positive results have not been experienced at all times, but there is no doubt that it is sometimes successful. The results obtained could be due to simple leaching of material from the cell, activity of endorespiratory enzymes within the living cell (79, 135), activity of autolytic enzymes in general within the cell following death (36, 139), or activity of specific autolytic ribonucleases from the cells themselves (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 conversion of pneumococci from a gram positive to a gram negative state following death, Avery and Cullen (3) found that they could isolate an autolytic enzyme which was active against pneumococci of all types, but not against other species of organisms. Later Dubos (51) showed that the active enzyme was a ribonuclease, and crude preparations of this enzyme were prepared. Bartholomew and Umbreit (13) took advantage of the newly available pure crystallized preparations of ribonuclease to show that this enzyme had a general ability to convert gram positive bacteria to a gram negative state. The work of Avery, Cullen, and Dubos was carried into extremely interesting phases by Jones, Stacey, and Webb (82) when they isolated species specific factors from Clostridium welchii and staphylococci. These factors, combined with the nucleinases of the cell, converted only one species of organism from gram positive to gram negative. The participation of ribonucleates in the gram positive state appears to be well established. Nevertheless, good evidence indicates that the gram positive state involves more than just the presence or absence of ribonucleates. It is important to note that other enzymes have also been shown to result in a conversion from the gram positive to the gram negative state. Webb (145) demonstrated this ability for lysozyme. Lysozyme was shown to act only on the polysaccharides or mucopolysaccharides of the cells; however, both polysaccharides and ribonucleic acids left the cell upon the action of the lysozyme. Webb suggested that both ribonucleic acids and polysaccharides are involved in the gram positive state. Others (148) have also reported the gram conversion power of lysozyme. Some workers (29) using very crude preparations reported that

13 1952] THE GRAM STAIN 13 such proteolytic enzymes as pepsin and trypsin could convert gram positive organisms to the gram negative state, but others (51, 82, 145) have been unable to confirm this observation for trypsin. The gram conversion of organisms by chemical pretreatment is less spectacular than the use of enzymes, and more confusing. It is often difficult to separate the action of the chemicals used from the action of autolytic enzymes normally present in the cells and from the action of nonspecific factors such as the 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 successive application of acid alcohol, ether, and benzyl chloride (61, 121, 136). Trommsdorff (139) reported that reversal could be accomplished if the cells were killed by chloroform but not if killed by boiling water. None of these reports is very helpful since the methods used, such as prolonged boiling with ether, could produce a multitude of changes within the cell. The same can be said for conversion by hydrolysis with HCl (144), strong acetic acid or alkalies (27), the conversion by 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 with some specific extractable material. Many workers have associated a change in gram character with the fatty components of the cell. Dreyer, Scott, and Walker (50) extracted staphylococci with ether and on their conversion from gram positive to gram negative isolated a lipoid from the extract. By ether extraction of gram positive bacteria Tamura (136) isolated a lipoid which he claimed was itself gram positive. Schumacher (121) isolated a fatty acid from yeast which according to his reports stained gram positively and which rendered protein gram positive. Other work has associated the gram positive to gram negative conversion with the nucleic acids of the cell. Deussen (47, 48, 49) used acid and alkaline hydrolysis to convert gram positive cells to gram negative. On conversion he 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 C for 12 to 144 hours and on conversion to the gram negative state noted that magnesium ribonucleate was the principal substance released from the cells. Some polysaccharide and protein were also observed. The polysaccharides have also been 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 presence of 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, the restoration to the gram positive state by these substances, and the reproducibility of the results (103). There is no doubt that ribonucleates are involved in the gram positive state, but it would be unwise to ignore completely the evidence

14 14 JAMES W. BARTHOLOMEW AND TOD MI'VrWER [VOL. 16 pointing to other factors such as lipoids, polysaccharides, and the cell membrane itself. 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 experiments are easily reproducible in a few minutes in any laboratory, and they present great difficulties to any strictly chemical concept of the gram positive state. However, as we will see later, the strictly chemical theories and the cell membrane permeability theories are not necessarily incompatible since permeability is undoubtedly associated with the chemical nature of the membrane. Other reported methods of gram conversion include the action of immune serum (114), the action of antibiotics (54), and mutation (133). A very simple conversion method is exposure to ultraviolet light of heat-fixed smears (Bartholomew and Mittwer, forthcoming publication). 3. Addition of gram positivity to cells previously rendered gram negative. The most outstanding work on the chemical nature of the gram positive state has been the demonstration that the gram negative cells produced by the removal of ribonucleic acids could be returned to the gram positive state by the addition of 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 then restored the gram positive state by exposure of the cells to ribonucleic acid. Henry and Stacey (68) removed a magnesium salt of ribonucleic acid from the cells of C. welchii and Saccharomyces cerevisiae, thus, converting these cells to the gram negative state. They then showed that magnesium ribonucleate from any source could restore the gram positive state to the converted cells provided these cells were kept in a reducing medium. Desoxyribonucleic acids, nucleosides, or nucleotides did not restore gram positivity, and the sodium salt of ribonucleic acids had but weak action. This observation was soon extended to a variety of species of microorganisms (69) and was soon confirmed in various aspects by other investigators (13). Magnesium ribonucleate failed, however, to make truly gram negative organisms such as E. coli gram positive (4, 7) although some success was obtained for an obviously intermediate organism such as members of the genus Neisseria. Thus, it is obvious that the cells must be capable of receiving the magnesium ribonucleate in a certain manner in order to create the gram positive state. Henry and his collaborators (70) have suggested that the cause of the gram positive state is the combination of magnesium ribonucleate with certain basic and novel types of proteins present only in gram positive cells. Webb (145) found that cells which were converted to the gram negative state by lysozyme could not be rendered gram positive by exposure to magnesium ribonucleate alone. This suggests that polysaccharides are part of the gram positive complex. 4. Conversion of cells normally gram negative to the gram positive state. Even normally gram negative bacteria such as E. coli and Aerobacter aerogenes have been reported to stain gram positively when grown on butter agar (29) or exposed to lecithin (50) or petrolatum (74). This work lacks conviction since the authors themselves report irregularities in results obtained. Bartholomew (7)

15 19521 THE GRAM STAIN 15 reported success in converting Neisseria but not E. coli into a gram positive state with magnesium ribonucleate; however, irregularities in results occurred. Baker and Bloom (4) reported that a viscous preparation of desoxyribonucleic acid caused E. coli to stain gram positively, but this gram positive state could easily be removed by washing with water. At present, it appears that there is no dependable way to convert normally gram negative cells to a gram positive state. Those agents which are successful in restoring the gram positive state to normally gram positive cells do not affect normally gram negative cells. This suggests again that the fundamental differences between the normally gram positive and gram negative types of cells are more than the simple presence or absence of such 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 staining procedure was also accompanied by a widespread interest in the reasons for the differentiation observed. Despite the efforts of many investigators, none has yet been able to describe definitively the mechanism although numerous interesting and significant experimental facts have been presented. Not only is it certain that the gram mechanism is much more complex than was at first believed, but it is possible that gram positive substrates vary as to the mechanism responsible 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 chemical theories which seek to show that gram positivity is a character attributable to some specific chemical substance or chemical complex within the cell. Second, the isoelectric point concept, which attributes a greater degree of dye-retention by gram positive organisms to the more acid state, e.g., lower isoelectric point, of their protoplasm. Third, the permeability theories, which explain gram positivity on the basis of particular permeability characteristics of the cell or cell membrane to such substances as alcohol, iodine, or the dye-iodine precipitate. Churchman's concept, associating gram positivity with the presence of a gram positive cell cortex (32, 33, 36), might also be referred to as an additional theory. However, since Churchman made no attempt to explain the gram positive state of the cortex, we shall discuss this concept in a following section concerning the site of the gram reaction. 1. Chemical theories of gram positivity. One of the earliest chemical theories was that proposed by Unna (142), who stated that an alcohol resistant dyeiodine-cell complex was formed in gram positive organisms, but not in gram negatives. This simple theory, however, offered no proof of the real existence, or the chemical 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-iodineunsaturated fatty acid complex which was resistant to dissociation by the action

16 16 JAMES W. BARTHOLOMEW AND TOD MITMWER [VOL. 16 of alcohol. Schumacher (121) reported the isolation of a gram positive fatty acid from yeast. The removal of this fatty acid by hydrolysis in acid alcohol rendered the yeast cell gram negative, and the gram positive state could be restored to this cell by exposure to an alcoholic solution of the cell substances removed. Thus, many of the earlier workers looked hopefully for an explanation of gram positivity 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 and Clostridium 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 gram positive cells contained unusual amounts of unsaturated fatty acids. Breinl (19) reported that, in general, gram negative cells took up more iodine than gram positives. Furthermore, Williams, Bloor, and Sandholzer (149) showed that nine strains of gram negative enteric bacilli contained considerable amounts of unsaturated fatty acids. Thus the role of unsaturated fatty acids in the gram positive 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 extract which stained gram positively (136). Generally, however, the work involving lecithin and lipoid extracts as the gram positive material has fared no better in its reproducibility than the work with the fatty acids. It has proved difficult or impossible for some workers to convert gram positive organisms to a gram negative 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 positive state 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" substances 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 reproducible in the hands of various workers. Deussen (47, 48, 49) first presented extensive experiments which showed that acid or basic hydrolysis of gram positive cells converted them into a gram negative state. He also showed that gram positivity could be restored by reintroducing nucleic acids into the extracted cells. Later Henry and Stacey (68, 69) converted cells to a gram negative state by the action of bile salts and observed that the substances given off during this conversion were magnesium ribonucleate, inert polysaccharides, and traces of protein. They also found that of the substances given off, magnesium ribonucleate could best restore gram positivity to the cells. Bartholomew and Umbreit (13) first used specific crystalline enzymes to show that magnesium ribonucleate played a definite role in gram positivity. Following the lead of Henry and Stacey, they found that crystalline ribonuclease converted all types of gram positive bacteria into the gram negative state and also confirmed the ability of magnesium ribonucleate to restore gram positivity to these cells. However, gram positivity is not due entirely to the presence or absence of ribonucleates since these have been demonstrated in gram negative forms, and it has been shown that gram

17 1952] THE GRAM STAIN 17 positive organisms are converted to a gram negative state even though only a fraction of the ribonucleic acids is removed from the cell (70, 81, 93, 105). It has also been shown that ribonucleic acids from gram negative organisms such as E. coli can be used to restore gram positivity to organisms such as C. welchii which have previously been rendered gram negative by the action of bile salts (81). Henry and his collaborators (68, 69, 70) have suggested that gram positivity involves the combination of ribonucleates with proteins of a very basic and novel type, and have reported the isolation of a gram positive nucleoprotein from gram positive organisms while they could not isolate it from gram negative organisms (70). This nucleoprotein could be hydrolyzed into gram negative nucleic acids and a gram negative protein, and its gram positivity restored by a recombination into a nucleoprotein. Stacey (130) has suggested that another contributing factor to gram positivity might be the ratio of ribonucleic acid (RNA) to desoxyribose nucleic acid (DNA). In C. welchii and the streptococci the RNA/DNA ratio was about 8.1 (70) whereas that for some gram negative organisms was reported as 1.3 (130). However, this concept of ratio was not well 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 to some objection unless a clear statement of the gram staining method used is given. This is true since the decolorization times of all stainable material are bound to vary, and thus many substances may appear to be gram positive, but only in comparison with very easily decolorized substances. Henry, Stacey, and Teece (70) made no comparison of the gram positive nature of their cell-free substances with such controls as Neisseria, E. coli, and yeast. It would seem that the reports of cell-free gram positive substances should be accepted only with reservations until much additional work has been done. Mitchell and Moyle (105) have suggested another possibility as to the nature of the gram positive substance. They found that determining nucleic acids in protein by analysis of the phosphate present sometimes gave much higher values than when the nucleic acids were determined by a pentose method. They showed that for M. pyogenes var. aureus the phosphate method gave a phosphate content of 30% over that accountable for on the basis of tetranucleotide structure. This excess was present mostly in the ribonucleic acid fraction, and Mitchell and Moyle attributed it to an unknown phosphoric ester "XP". Mitchell and Moyle then suggested that there was a direct correlation between this "XP" ester and the gram positive state. They backed this claim with an analysis of 16 different organisms. Their data do show a general correlation between "XP" ester and gram positivity but become less convincing on an examination of details. For instance, if "XP" ester is responsible for gram positivity, it should be present in all gram positive organisms, but the data of Mitchell and Moyle show it to be absent in C. welchii. Also, some unmistakably gram negative organisms such as E. coli, A. aerogenes, and Neisseria catarrhalis contained as much or 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 18 JAMES W. BARTHOLOMEW AND TOD MIX'rWER [VOL. 16 for gram positivity, but little evidence exists as yet that it is the gram positive substance. 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 different from the gram positive substance of Henry and Stacey since it was an acid protein of high nitrogen content and was not a nucleoprotein. The gram staining procedure was not given in this report, and as in other cases of extracellular gram positivity which have been reported, the reader is left uncertain as to the degree 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 a gram positive substance in themselves, nor is there reason to believe that they constitute the sole factor producing gram positivity in the cell. Polysaccharide material has also been associated with gram positivity (68, 145), but as yet its role has not been experimentally determined. 2. The isoelectric point concept. Steam and Steam (132, 134) proposed a different concept of the relationship between cell chemistry and the gram positive characteristic. They did not seek a gram positive substance but sought to correlate the over-all isoelectric point (or isoelectric range) of the cell substance with the gram staining properties of the cell. They determined isoelectric points on the basis of cell affinity for acid and basic dyes at various ph levels. Their isoelectric point was the ph at which the cell showed equal affinity for both acid and basic dyes. They showed that increasing amounts of basic dyes were taken up as the ph increased over the isoelectric point, and increased amounts of acid dyes were taken up as the ph decreased from the isoelectric point. Using this method, they reported that gram positive organisms generally possessed isoelectric points somewhat below those of gram negative organisms. Steam and Steam reasoned that since the gram stain is carried out at a ph near 7, the gram positive cells would take up more dye than the gram negative and that they also would retain the dye with greater tenacity. Hence, the gram negative cells would be easier to decolorize during the alcohol step. Their concept of the function of iodine was that it simply increased the range of the differences between the isoelectric points of the gram positive and gram negative cells, thus making the differentiation more marked. They believed that the action of iodine was to oxidize certain substances in gram positive cells, thus rendering them more acidic and lowering their isoelectric point, and that any oxidizing agent would have the same effect as iodine and could be substituted for it. This concept sounds logical in many respects, but it encounters serious difficulty in view of several known experimental facts. In the first place, many workers have not been able to confirm Steam and Steam's contention that any oxidizing 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 differentiation even though the iodine is applied before the dye, and this has been shown not to be true (26, 27, 64, 106). Also, if this theory were correct, it should be

19 19521 THE GRAM STAIN 19 applicable to crushed cells as well as intact cells, but as has been pointed out earlier, crushed cells do not stain gram positively. Levine (99) showed that the methods used by Steam and Steam to determine isoelectric points were incapable of actually determining a true isoelectric point, and that results sometimes varied more than two ph units. Lastly, Steam and Stearn did not use a counterstain 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's work was valuable in that it contributed to general knowledge of staining phenomena; however, their conclusions regarding the mechanism of gram differentiation can hardly be accepted in the light of more recent knowledge. 3. Theories involving permeability. The possibility that gram differentiation could be due to differences in the permeability of the cell as a whole, or of the cell membrane, has always received considerable consideration. These theories have been objected to on the basis that it is problematic that a killed cell retains any selective permeability characteristics (45). However, the suggested permeability differences are not necessarily of the selective type. The differences might be simply in permeability to substances in general. Fischer (58) believed that the cell substance of gram positive bacteria is less permeable 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 did conduct some experiments designed to show that the protoplasm of gram positive bacteria consisted of coarser granules than that of gram negative bacteria. Brudny (21) expanded the concept of Fischer and proposed that the difference between gram positive and gram negative bacteria was permeability of the cell as a whole to iodine. He believed that a dye-iodine precipitate was formed in the interstitial 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 the interstitial spaces. Thus, the dye-iodine precipitate was formed on the outside of the cell and was easy to remove. The specific concepts of Fischer and Brudny did not solve the problem of the mechanism of gram differentiation. However, their general idea of the significance of permeability is becoming accepted more and 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 cell membrane. His experiments were impressive in that they showed that a crushed gram positive cell always stained gram negatively. This experimental fact is easily reproducible (26, 27, 106, 125) and must be considered by anyone advocating a strict chemical concept of gram positivity. Benians presented good evidence against a strictly dye-iodine-cell substance complex when he showed that stained cell debris actually decolorized easier after iodine treatment than before. In his first paper (16) he theorized that iodine rendered the membranes of gram positive bacteria impermeable to alcohol, but he presented no direct proof to substantiate this idea. In his later paper (17) he had altered his views

20 20 JAMES W. BARTHOLOMEW AND TOD M17TWER [VOL. 16 and divided all bacteria into the following three types: First, the regular gram positive organisms in which the dye and iodine penetrate into the cell and there form a large molecule which does not easily pass out of the cell during decolorization. Second, the regular gram negative organisms in which the primary dye does 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 and iodine penetrated into the cell, but the dye-iodine precipitate easily passed out of 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 increase its size sufficiently to influence its permeability. Benians based his conclusion that the primary dye does not enter gram negative cells on the observation that cells of E. coli were colorless when centrifuged down from a suspension in methyl violet. He ignored the fact that E. coli readily takes the stain on a heat fixed slide. Benians' work is impressive mostly for his observation on the effect of crushing the cell, and that iodine did not fix dye to cell protein, but rather detaches it therefrom, thus explaining Gram's original observation that gram negative 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 gram reaction. Their experiments showed that iodine in the decolorizer had a drastic retarding effect on decolorization. They concluded that two factors contributed to gram positivity. One was the low alcohol solubility of the dye-iodine compound, and the other was the lower permeability of gram positive cells to iodine in alcoholic solution. Thus, on decolorization the dye-iodine complex would be much slower in dissolution and in leaving the cell in gram positive bacteria. This concept was given additional weight by the observation of Mittwer, Bartholomew, and Kallman (106) that gram positive cells took up alcoholic iodine more slowly than gram negative cells, and that only dye-iodine precipitates with certain 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 a gram positive external cortex layer which surrounded a gram negative internal medulla. He supported this concept by showing that acriviolet conversion of Bacillus anthracis to a gram negative state was accompanied by a 40% reduction in cell size as measured by the filar micrometer. This size difference was obvious even upon visual examination of his preparations under the microscope. Similar visual evidence could also be seen when yeast was converted to a gram negative state by holding in water suspensions at high temperatures. In addition, Churchman reported a 50% weight loss when cocci were converted to a gram negative state. The validity of Churchman's concepts has never really been proved or disproved. Certainly everyone who has studied gram stains has noticed occasional small thin gram negatively staining cells in a chain of larger gram positively

21 1952] THE GRAM STAIN 21 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 exposure to ribonuclease, without an apparent size reduction (11, 135). Therefore, Churchman's cortex itself could not be the cause of gram positivity since it can be made gram negative by the loss of chemical substances. Other workers have confirmed that reduction of cell size may occur when gram positive cells are converted to a gram negative state (79, 106), but this size reduction may follow by some hours the change in gram staining character (106). No proof is available to show that Churchman's medulla is gram negative when surrounded by the gram positive cortex, although it is gram negative when the cortex is removed. Henry, Stacey, and Teece (70) have presented pictures of yeast partially converted to the gram negative state by the action of bile salts. These pictures suggest that an area analogous to Churchman's medulla is still strongly gram positive 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 of gram 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 "ectoplasm" layer simply by a staining technique. Churchman disclaims any connection, but Gutstein reported that this ectoplasm layer disappeared when yeast 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 gram negative 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 gram positive material is located in an outer cortex area of the cell and that the presence or absence of this cortex is directly associated with the gram positive characteristic. Gutstein's work suggests that the gram positive material is associated with the ectoplasm area, but not necessarily identical with it, since gram negative cells also possessed this area. Lamanna and Mallette (93) stated that the gram positive material included the cell wall as well as the cell cytoplasm, a conclusion based on the observations that adjacent yeast cells could be shown to be in contact by both the gram and a cell wall stain. They also observed that in chains of gram positive cells containing an occasional gram negative cell the gram negative cells took the cell wall stain whereas the cell wall area of 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 same cells, stained successively by gram and a cell wall stain, they found that the gram stained cell could be fitted within the area left unstained by the cell wall method. Cell measurements confirmed this concept except for a small area of overlapping. 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 thickness. He found that all sectioned cells stained gram negatively so he could not

22 22 JAMES W. BARTHOLOMEW AND TOD MITTWER [VOL. 16 observe the site of gram positive material. Bartholomew (6) applied the new methods of cutting thin sections of 0.05 ;i, but few definite conclusions could be obtained from the sections. It appears then that the site of the gram reaction is still in doubt and it can be spoken of only in general terms. Indications are that the gram positive area is certainly throughout the interior of the cell, and that it may include the cell wall either completely or partially. VII. CONCLUSIONS In summary, the gram positive state cannot yet be ascribed to any one unique chemical compound or complex present in gram positive, but absent in gram negative organisms. This is true even though several compounds have been shown to be closely associated with the gram positive state. It also seems that cell membrane permeability must be given an important role since crushed or mutilated cells always stain gram negatively. It is probable that chemical composition 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 solely responsible for gram positivity; especially since removal of a number of very different compounds has been shown to result in loss of gram positivity; these include magnesium ribonucleate, lipids, polysaccharides, fatty acids, nucleoproteins, and unknown phosphoric esters. Likewise, the list of reagents or processes which convert cells to a gram negative state is long: water, ribonucleases, lysozyme, boiling ether, fat solvents, acids, alkalies, acriviolet dye, oxidizing agents, bile salts, crushing, immune serum, antibiotics, ultraviolet radiation. It is likely that removal of lipoids, ribonucleates, polysaccharides, or other compounds results in a more permeable cell membrane through the disruption of its molecular 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 differentiation: Primary stain. Gram positive and gram negative cells take up the primary stain by an ionic bond between the basic groups of the dye and the acidic groups of the cell (8). It is possible that both types of cells take up approximately equal amounts of dye (105). Iodine. The iodine, in aqueous solution, enters both types of cells. Here, it forms a precipitate with the dye present, either by competitive removal of the dye from the protein, or by addition to the dye in situ. Alcohol. It is in this step that the actual differentiation takes place. In gram negative cells the alcohol freely passes the cell membrane, dissolves and dissociates the dye-iodine precipitate or complex, and washes it away, leaving the cell colorless. In gram positive cells the alcohol, the iodine in alcoholic solution, or both pass through the membrane only with difficulty. Consequently, dissolution of 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

23 19521 THE GRAM STAIN 23 is left in the cell and the cell retains its color. It should be emphasized that this permeability characteristic varies greatly on a wide scale, resulting in decolorization times of a few minutes to many hours. All gram positive cells will decolorize if exposed long enough to alcohol. If various substances such as magnesium ribonucleate, mucopolysaccharides, or lipoproteins are removed from the cell membrane, the latter becomes more permeable to the decolorizer, and the cell reacts to the gram stain in the same manner as do normal gram negative cells. Counterstain. The counterstain freely stains the gram negative cells which have been restored in the alcohol step to their original colorless condition. For practical gram staining the reviewers wish to quote and emphasize the recommendation of Hucker and Conn (77) that "to determine the tendency of an organism with regard to the gram stain, more than one staining procedure be used, and that preparations of the culture be prepared at various stages of growth." Also, as recommended by Burke (24), control organisms should be placed on the same slide to determine the validity of the gram stain. It is our opinion that before a substance be called gram positive it should be able to withstand a decolorization step sufficient to render gram negative the Neisseria species and also the nuclei of plant and animal cells as these two substances have been commonly accepted as being gram negative. There is an enormous literature on the gram stain, and an immense amount of research has been performed to elucidate its mechanism. More will undoubtedly be done in the future. Much misinterpretation of results and much needless duplication 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 depending in whole or in part on the gram stain describe their staining method in considerable detail... their results will have more significance in other quarters if the exact steps of the staining procedure are published." VIII. REFERENCES 1. ALBERT, R. AND ALBERT, W Chemische VergAnge in der abgetbteten Hefezellen. Zentr. Bakt. Parasitenk., Abt. II, 7, ATKINS, K. N Report of committee on descriptive chart. Part III. A modification of the gram stain. J. Bact., 5, AVERY, 0. T. AND CULLEN, G. E Studies on the enzymes of pneumococcus. IV. Bacteriolytic enzymes. J. Exp. Med., 38, BAKER, H. AND BLOOM, W. L Further studies on the gram stain. J. Bact., 56, BAKER, Z., HARRISON, R. W., AND MILLER, B. F The bactericidal action of synthetic detergents. J. Exp. Med., 74, BARTHOLOMEW, J. W. Unpublished data. 7. BARTHOLOMEW, J. W Gram positive characteristics of the Neisseria. J. Bact., 51, BARTHOLOMEW, J. W., EVANS, E. E., AND NIELSEN, E. D The effect of esterification of protein carboxyl groups on the staining of bacterial cells. J. Bact., 58, BARTHOLOMEW, J. W. AND MITTWER, T The mechanism of the gram reaction. I. The specificity of the primary dye. Stain Technol., 25,

24 24 JAMES W. BARTHOLOMEW AND TOD MITTWER [VOL BARTHOLOMEW, J. W. AND MITTWER, T The mechanism of the gram reaction. III. Solubilities of dye-iodine precipitates and further studies of primary dye substitutes. Stain Technol., 26, BARTHOLOMEW, J. W. AND MITTWER, T Cell structure in relation to the gram reaction as shown during lysis of Bacillus subtilis. J. Gen. Microbiol., 6, BARTHOLOMEW, J. W., ROBERTS, M. A., AND EVANS, E. E Dye exchange in bacterial cells, and the theory of staining. Stain Technol., 25, BARTHOLOMEW, J. W. AND UMBREIT, W. W Ribonucleic acid and the gram stain. J. Bact., 48, BtGUET, M Sur les limites de la spkcificitm de la reaction de Gram. Compt. rend. soc. biol., 101, BtGUET, M Sur le m6canisme de la m6thode de Gram. Compt. rend. soc. biol., 101, BENIANS, T. H. C Observations on the gram positive and acid fast properties of bacteria. J. Path. Bact., 17, BENIANS, T. H. C A further investigation into the principles underlying Gram's stain, with special reference to the bacterial cell membrane. J. Path. Bact., 23, BENOIT, M Recherches sur la r6action de Gram. Nancy. Socidt6 d'impressions Typographiques, 134 pp. 19. BREINL, F tuber das Verhalten grampositiver und gramnegativer Bakterien zu den Halogenen. Z. Immunitatsforsch., 29, BRICE, A. T., JR Three notes on biological stains. Am. J. Clin. Path., 3, BRUDNY, V tmber die Beziehung zwischen der FArbarkeit der Bakterien nach Gram und ihrer Permeabilitat. Zentr. Bakt. Parasitenk., Abt. II, 21, BRUNER, D. W Differentiation between gram positive and gram negative microorganisms by the use of enzymes. J. Bact., 26, BUGGS, C. W. AND GREEN, R. G Electrophoretic phenomena of bacteria. III. The electrophoretic velocity of bacteria in relation to growth, senescence, and death. J. Bact., 30, BURKE, V Notes on the gram stain with description of a new method. J. Bact., 7, BURKE, V. AND ASHENFELTER, M Notes on the Gram stain. Stain Technol., 1, BURKE, V. AND BARNES, M The cell wall as a factor in the gram reaction. J. Bact., 15, BURKE, V. AND BARNES, M. W The cell wall and the gram reaction. J. Bact., 18, BURKE, V. AND GIBSON, F The gram reaction and the electric charge of bacteria. J. Bact., 26, CEDERCREUTZ, A Studien fiber die Bedingungen des positives oder negativen Ausfallens der Gramf~rbung bei einigen Bakterien. Arch. Dermatol. u. Syphilis, 93, CHRISTENSEN, W. B Observations on the staining of Corynebacterium diphtheriae. Stain Technol., 24, CHURCHMAN, J. W The selective bactericidal action of gentian violet. J. Exp. Med., 16, CHURCHMAN, J. W The structure of B. anthracis and the reversal of the gram reaction. Proc. Soc. Exp. Biol. Med., 24, CHURCHMAN, J. W The structure of B. anthracis and the reversal of the gram reaction. J. Exp. Med., 46, CHURCHMAN, J. W Effect of hardening and fixation on gram reaction. Proc. Soc. Exp. Biol. Med., 25,

25 19521 THE GRAM STAIN CHURCHMAN, J. W Staining Reactions of Bacteria. In The Newer Knowledge of Bacteriology and Immunity, by Jordan, E. 0. and Falk, I. S., Chicago. Refer to pp CHURCHMAN, J. W Gram structure of cocci. J. Bact., 18, CHURCHMAN, J. W. AND SIEGEL, L Cultural separation of bacteria on the basis of triphenylmethane coefficients. Stain Technol., 3, CONN, H. J An investigation of American gentian violets. J. Bact., 7, CONN, H. J Variability of the gram reaction. Stain Technol., 2, CONN, H. J A new substitute for ethyl alcohol in the gram stain. Stain Technol., 3, COUDRAY, J Contribution A l'6tude de la coloration de Gram. These doct. Univ. Nancy, 112 pp. 42. COWLES, P. B Alkyl sulfates: their selective bacteriostatic action. Yale J. Biol. and Med., 11, DAY, A. A. AND GIBBS, W. M The action of pancreatic juice on bacteria. J. Infectious Diseases, 46, DEBoRD, G. G Reaction to Gram's stain by certain spore-forming bacteria. Proc. Soc. Exp. Biol. Med., 22, DELUTANG, R Contribution a l'6tude de la reaction de Gram. Les Presses Modernes, Paris, 134 pp. 46. DELEY, J Cytochemical observations on nucleic acids and nucleoproteins in nitrogen-deficient microorganisms. Antonie van Leeuwenhoek J. Microbiol. Serol., 17, DEUSSEN, E Die Gramsche Bakterienfarbung, ihr Wesen und ihre Bedeutung. Z. Hyg. Infektionskrankh., 85, DEUSsEN, E Die Gramsche Bakterienfarbung, ihr Wesen und ihre Bedeutung. Biochem. Z., 103, DEUSSEN, E Die Gramsche BakterienfArbung, ihr Wesen und ihre Bedeutung. Z. Hyg. Infektionskrankh., 93, DREYER, G., ScoTT, S. G., AND WALKER, E. W. A Preliminary note on the nature of gram staining. J. Path. Bact., 16, DUBOS, R. J The autolytic system of pneumococci. J. Exp. Med., 65, DuBos, R. J Microbiology. Ann. Rev. Biochem., 11, DUIBos, R. J Inhibition of bacterial growth by auxins. Proc. Soc. Exp. Biol. Med., 63, DUFRENOY, J. AND PRATT, R Cytochemical mechanisms of penicillin action. III. Effect on reaction to the gram stain in Staphylococcus aureus. J. Bact., 54, EISENBERG, P Studien zur Ektoplasmatheorie. IV. Zur Theorie der Gramfestigkeit. Zentr. Bakt. Parasitenk., Abt. I, Orig., 56, EISENBERG, P Untersuchungen uiber die Variabilitat der Bakterien. VII Mitt. tber die Variabilitat des Schleimbildungsvermogens und der Gramfestigkeit. Zentr. Bakt. Parasitenk., Abt. I, Orig., 82, ERNST, P Ein Verhornender Plattenepithelkrebs des Bronchus: Metaplasie oder Aberration? Beitr. path. anat. u. allgem. Path., 20, FISCHER, A Vorlesungen uber Bakterien, Jena. Translated into English by A. Coppen Jones: The structure and functions of bacteria. Oxford, The Clarendon Press, FRIEDLANDER, C Die Mikrokokken der Pneumonie. Fortschr. Med., 1, GALE, E. F. AND RODWELL, A. W Amino acid metabolism of penicillin resistant staphylococcus. J. Bact., 55, GOADBY, K Bacterial proteins. The presence of alcohol-soluble proteins in bacteria. Proc. Roy. Soc. (London), B, 102,

26 26 JAMES W. BARTHOLOMEW AND TOD MITTWER [VOL GRAHAM-SMITH, G. S Some factors influencing the actions of dyes and allied compounds on bacteria. J. Hyg., 18, GRAM, C tuber die isolirte Ffirbung der Schizomyceten in Schnitt- und Trockenpraparaten. Fortschr. Med., 2, GUERBET, M., MAYER, A., AND SCHAEFFER, G Sur les r6actions microchimiques des corps gras et la r6action de Gram. Compt. rend. soc. biol., 62, GUTSTEIN, M Das Ektoplasma der Bakterien. II. t~ber farberische Verscheidenheiten zwischen grampositiven und gramnegativen Bakterien. Ein Beitrag zur Theorie der Gramschen FArbung. Zentr. Bakt. Parasitenk., Abt. I, Orig., 94, GUTSTEIN, M Das Ektoplasma der Bakterien. V. FArberischer Nachweis und chemischer Bau des Ektoplasmas der Gramnegativen Bakterien. Zentr. Bakt. Parasitenk., Abt. I, Orig., 100, 1-9. / 67. HENRICI, A. T The staining of yeasts by Gram's method. J. Med. Research, 30, HENRY, H. AND STACEY, M Histochemistry of the gram staining reaction for microorganisms. Nature, 151, HENRY, H. AND STACEY, M Histochemistry of the gram staining reaction for microorganisms. Proc. Roy. Soc. (London), B, 133, HENRY, H., STACEY, M., AND TEECE, E. G Nature of the gram positive complex in microorganisms. Nature, 156, HENRY, R. J The mode of action of sulfonamides. Bact. Revs., 7, HIJMANNS VAN DEN BERG, A Uber das Verhalten des Gonococcus zur Gramschen Farbemethode. Zentr. Bakt. Parasitenk., Abt. I, 20, HOLLBORN, K Nachtrag zu meiner Arbeit "Zwei neue simultanfarbstoffe". Zentr. Bakt. Parasitenk., Abt. I, Orig., 139, HOLMAN, W. L An error in acid-fast and gram staining due to petrolatum. Arch. Path. Lab. Med., 1, HUCKER, G. J A new modification and application of the gram stain. J. Bact., 6, HUCKER, G. J Comparison of various methods of gram staining (preliminary report). Abstracts Bact., 6, HUCKER, G. J. AND CONN, H. J Further studies on the methods of gram staining. N. Y. (Geneva) Agr. Exp. Sta. Tech. Bull., 128, HUNTOON, F. M Glycerine as an adjuvant to bacterial dyes. Am. J. Clin. Path., 1, INGRAM, M The endogenous respiration of Bacillus cereus. I. Changes in the rate of respiration with the passage of time. J. Bact., 38, JOBLING, J. W. AND PETERSON, W Bacterial anti-ferments. Studies on fermentation. J. Exp. Med., 20, JONES, A. S., MUGGLESTON, P. W., AND STACEY, M The gram complex in Clostridium welchii. Nature, 166, JONES, A. S., STACEY, M., AND WEBB, M Studies on the autolytic systems of gram positive microorganisms. I. The lytic system of staphylococci. Biochim. et Biophys. Acta, 3, KAPLAN, M. L. AND KAPLAN, L The gram stain and differential staining. J. Bact., 25, a. KEMP, H. A. AND FLEISCHER, M. S The bactericidal influence of various substances upon gram positive and gram negative bacteria. Proc. Soc. Exp. Biol. Med., 24, KISSKALT, C Eine Modifikation der Gramschen Ffarbung. Zentr. Bakt. Parasitenk., Abt. I, 30, KLIGLER, I. J A study of the antiseptic properties of certain organic compounds. J. Exp. Med., 27,

27 19521 THE GRAM STAIN KNAYSI, G Elements of Bacterial Cytology. Comstock Publishing Co., Ithaca, N. Y., 209 pp. 87. KNAYSI, G., HILLIER, J., AND FABRICANT, C The cytology of an avian strain of Mycobacterium tuberculosis studied with the electron and light microscopes. J. Bact., 60, KNOTHE, H. AND MAASEN, W tvber eine Modifikation der Gram-farbung mit Borsaure als Vorbeize und Pikrinsiiure-Sublimat-Natriumacetat-Wasser-Gemisch statt Lugolscher Losung als Hauptbeize. Z. Hyg. Infektionskrankh., 128, KOPELOFF, N. AND BEERMAN, P Modified gram stains. J. Infectious Diseases, 31, KOPELOFF, N. AND COHEN, P Further studies on a modification of the gram stain. J. Bact., 15, KRAJIAN, A. A A new and rapid staining method for gram positive and gram negative organisms in frozen and paraffin sections. J. Lab. Clin. Med., 28, KRETSCHMER, 0. S The gram property of the acid fast form of the tubercle bacillus. J. Lab. Clin. Med., 19, LAMANNA, C. AND MALLETTE, M. F The relation of the gram stain to the cell wall and the ribonucleic acid content of the cell. J. Bact., 60, LMRsON, W. P., HARTZELL, T. B., AND DIEHL, H. S The effect of high pressures on bacteria. J. Infectious Diseases, 22, LASSEUR, P. AND BENOIT, M Observations sur la m6thode de Gram. Trav. Lab. Microbiol. facult6 pharm. Nancy, 7, LASSEUR, P., DUPmx, A., AND MAGNITOT, C Application du ph6nomene de Boutaric A la preparation des solution colorantes. Trav. Lab. Microbiol. facult6 pharm. Nancy, 4, LASSEUR, P., DUPAIX-LASSEUR, A., AND FRIBOURG, R Transport 6lectrique des bact6ries. Trav. Lab. Microbiol. facult6 pharm. Nancy, 7, LASSEUR, P. AND SCHMITT 1927 etude de la r6action de Gram. Ann. inst. Pasteur, 41, LEVINE, N. D The determination of apparent isoelectric points of cell structures by staining at controlled reactions. Stain Technol., 15, LILLIE, R. D The gram stain. I. A quick method for staining gram positive organisms in tissues. Arch. Path., 5, LILLE, R. D The gram stain. II. Resistance of the tuberculosis-leprosy group of organisms to decolorization with sodium thiosulfate. Arch. Path., 5, MARCHAL, J. G Reaction de Gram. Facteurs influengant la durde de ddcoloration. Bull. Assoc. dipl6m6s Microbiol. facult6 pharm. Nancy, 19, MARCHAL, J. G. AND NICOT, R Observations sur la methode de Gram appliqu6e A des bact~ries (Gram positive) apr~s action de la ribonucldase. Compt. rend. soc. biol., 143, MEYRICK, L. D. AND HARRISON, C. V A counterstain for use in gram technique. J. Path. Bact., 54, MITCHELL, P. AND MOYLE, J Occurrence of a phosphoric ester in certain bacteria: its relation to gram staining and penicillin sensitivity. Nature, 166, MITrWER, T., BARTHOLOMEW, J. W., AND KALLMAN, B. J The mechanism of the gram reaction. II. The function of iodine in the gram stain. Stain Technol., 25, MUDD, S. AND SMITH, A. G Electron and light microscope studies of bacterial nuclei. I. Adaptation of cytological processing to electron microscopy; bacterial nuclei as vesicular structures. J. Bact., 59, NEIDE, E Die Alkoholentfirbung der nach Gram gefarbten Bakterien als

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