Bactrev00008 0007

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 ....................................................... 2
1. The primary stain ....................................................... 3
2. The mordant ............................................................ 4
3. The decolorizer ......................................................... 4
4. The counterstain ........................................................ 5
5. Other factors ............................................................ 6
III. CORRELATION OF GRAM REACTION WITH PHYSIOLOGICAL CHARACTERISTICS ...... 8
IV. DEGREE OF GRAM POSITIVITY . ............................................... 10
1. Studies of decolorization time. Gram Dauer character.10
2. Conversion of gram positive cells to gram negative .12
3. Addition of gram positivity to cells previously rendered gram negative... 14
4. Conversion of cells normally gram negative to the gram positive state.... 14
V. THE MECHANISM OF GRAM DIFFERENTIATION . .15
1. Chemical theories of gram positivity .15
2. The isoelectric point concept. ...................................... 18
3. Theories involving permeability ...................................... 19
VI. THE SITE OF THE GRAM REACTION.20
VII. CONCLUSIONS. ............................................................... 22
VIII. REFERENCES. ................................................................ 23
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 micro-
cocci 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 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 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 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 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 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 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 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 valua-
ble 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 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 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 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 out
of 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 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-
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 repro-
ducible 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 com-
monly 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 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 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 sugges-
tions 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 addi-
tion, 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.
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 iodine-
potassium 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 Con-
stable (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. 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 little
practical 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 a
way that the alcohol does not change its dilution. Blotting dry before decoloriza-
tion 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 al-
cohol. Conn (40) found a mixture of equal parts of methyl and isopropyl alcohols
to have very similar decolorization properties to ethyl alcohol. In practice, how-
ever, 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 depres-
sant (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 determina-
tions of the temperature effect are not now available, but it is established that
an increase in temperature results in more rapid decolorization (45). The evi-
dence 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
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 this
is so, then the factors influencing dye replacement assume importance in the
gram procedure. Since the replacement tendency is in proportion to the concen-
tration 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 de-
colorization. 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 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 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 indi-
cated 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 con-
taining 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
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 in-
fluence 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 cul-
tures 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
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 (potassium
dichromate, 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 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 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 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 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 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 ob-
jects. 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 con-
flicting 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-
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, 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 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
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 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 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 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 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 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 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) varia-
bility 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 intro-
duced 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-
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 modi-
fied 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). Varia-
tions 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 me-
dium, 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 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 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 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 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 nega-
tive. 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
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 con-
flicting 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 Um-
breit (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 staphylo-
cocci. 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. Never-
theless, 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 polysaccha-
rides 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
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 spec-
tacular 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 pro-
duce 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 posi-
tive 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 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 gram
positive. 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 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 mag-
nesium 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 reproducibil-
ity 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 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 mem-
brane 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 (Bartholo-
mew 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, nucleo-
sides, 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) al-
though 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 ribo-
nucleate 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 ex-
posed to lecithin (50) or petrolatum (74). This work lacks conviction since the
authors 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 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 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 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 in-
teresting and significant experimental facts have been presented. Not only is it
certain 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 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 precipi-
tate.
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 posi-
tive 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 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 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-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 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 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 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" 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 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 conver-
sion 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 ribo-
nucleate 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
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 posi-
tivity 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 or-
ganisms 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 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 pro-
tein 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 dif-
ferent concept of the relationship between cell chemistry and the gram positive
characteristic. They did not seek a gram positive substance but sought to cor-
relate 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 iso-
electric 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 diffi-
culty 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 differen-
tiation 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
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 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 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 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 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 per-
meability 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 posi-
tive 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 advo-
cating 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 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 decolor-
ization. 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 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 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 com-
pound, 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, Bar-
tholomew, 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% reduc-
tion 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 dis-
proved. Certainly everyone who has studied gram stains has noticed occasional
small 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 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 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 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 "ecto-
plasm" layer simply by a staining technique. Churchman disclaims any con-
nection, 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 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 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 cyto-
plasm, 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 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 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 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 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 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 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, 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, oxidizing
agents, 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 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 differentia-
tion:
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 disso-
ciates 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
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 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 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 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 depending
in 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 the
exact steps of the staining procedure are published."
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THE GRAM STAIN

JAMES W. BARTHOLOMEW AND TOD MITTWER

staining cells. This would appear to confirm Churchman's concepts. However,

35. CHURCHMAN, J. W. 1928 Staining Reactions of Bacteria. In The Newer Knowledge

86. KNAYSI, G. 1944 Elements of Bacterial Cytology. Comstock Publishing Co.,

Speciesdiagnose, in Verbindung mit einer Untersuchung der fur die Gramfarbung

132. STEARN, A. E. AND STEARN, E. W. 1926 Studies in the physico-chemical behavior

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