The Mechanism of the Gram Reaction. II. The Function of Iodine in the Gram Stain

Fifty-five reagents were studied as to their ability to replace iodine in the Gram stain. None gave results as good as iodine. Eight gave usable Gram preparations, and forty-seven gave negative results. Omission of the counterstain resulted in increasing to thirty-three the number of reagents giving differentiation, but this, was not considered a true Gram differentiation. Many oxidizing agents were shown not to be substitutes for iodine; therefore the function of iodine must be more than to serve as an oxidizing agent. Many reagents which formed precipitates with the dye could not replace iodine; therefore factors other than precipitate formation must be involved. However, all agents which were good substitutes for iodine were both good oxidizing and dye precipitating agents. Experiments involving the study of cell membrane permeability showed that Gram-positive cells were less permeable to iodine in alcoholic solution than Gram-negative cells. This difference could not be demonstrated for iodine in aqueous solution. It was concluded that iodine served to form a dye-iodine precipitate (or complex) in the cell. Since Gram-positive cells were less permeable to iodine in alcohol than Gram-negative cells, this resulted in a slower dissolving out of this complex from Gram-positive cells during de-colorization and hence a slower decolorization time. The relative solubilities of dye precipitates in alcohol and in aqueous safranin solution were also indicated as an important factor influencing decolorization. Dyes which formed highly soluble precipitates with iodine could not be used in the Gram stain. It is not proposed that the mechanism of the Gram stain is entirely one of membrane permeability; chemical factors are undoubtedly important and will be discussed in a later paper. However, it is proposed that the chemical and physical factors are closely interrelated in the Gram stain mechanism.     

The Phenomenon of Gram-Positivity; Its Definition and Some Negative Evidence on the Causative Role of Sulfhydryl Groups

It has been accepted for many decades that a Gram-positive organism is one which retains the primary dye when stained by accepted Gram stain procedures. It has also been known that the iodine step is essential if Gram differentiation is to be obtained. If bacterial cells are treated in such a way that they will retain the primary dye following a Gram staining procedure, regardless of whether or not the iodine step is included, then the mechanism of this dye retention must differ from that which normally is responsible for a Gram-positive state. Similarly, when both the iodine and decolorization steps are omitted, the counter-stain should always replace the primary stain. If it does not, then the mechanism of dye retention would not be normal, and any such dye retention would not be related to the Gram phenomenon. In such cases one is not studying the Gram reaction, but is studying chemical affinities or physical states which produce visually similar but actually unrelated phenomena. Failure to appreciate this has resulted in papers appearing under the guise of studies of the Gram reaction which have little or no relationship to the Gram phenomenon.

In the interest of consistency, these criteria of true Gram-positivity (the necessity of iodine for Gram-positivity with a normal Gram procedure, and the ability of the counterstain to replace the primary dye when both the iodine and decolorization steps are omitted) should be applied to both intact cells and cell-free substances, even though their mechanism of Gram-positivity may differ.

The above criteria have been applied in a study of the sulfhydryl concept of the mecharism of Gram-positivity as proposed by Fischer and Larose. It was found that while the experimental work of Fischer and Larose was reproducible, the supposedly Gram-positive states produced did not possess the characteristics which would identify them as true Gram-positive states. Our results would not support the sulfhydryl concept concerning the mechanism of Gram-positivity.     

The Mechanism of the Gram Reaction. III. Solubilities of Dye-Iodine Precipitates and Further Studies of Primary Dye Substitutes

Solubilities of dye-iodine precipitates in alcohol and in aqueous safranin solution were determined by direct solubility methods and by photocolorimetric methods. It was found that, increasing precipitate solubility in alcohol or safranin solution gave decreasing differentiation between Gram-positive and Gram-negative bacteria. Dyes which did not stain the cells well as a primary stain did not give good Gram stains, regardless of the solubilities of their precipitates. Some dyes (typified by methylene blue) which gave relatively alcohol-insoluble iodine precipitates gave inferior Gram differentiation because these precipitates were readily soluble in the safranin counterstain.

Solubilities of precipitates of crystal violet and various iodine substitutes were determined photocolorimetrically. The ability of a substance to replace iodine in the Gram stain correlated with its ability to give a precipitate which was only slightly soluble in alcohol and relatively insoluble in aqueous safranin solution.

It was concluded that the usual Gram reagents are not truly specific for the differentiation. Any dye and mordant could be used if the dye was deeply colored, stained the cells well, and if the precipitate of dye and mordant was only slightly soluble in alcohol and relatively insoluble in the counterstain. These factors, combined with those influencing differences in cell membrane permeability, constitute the most important factors in the Gram stain differentiation.

Studies were made concerning the ability of dyes to substitute for crystal violet in the Gram procedure. Of 29 dye samples reported on here for the first time none proved to be good substitutes for crystal violet.     

The Mechanism of the Gram Reaction I. The Specificity of the Primary Dye

Dyes of all major types were tested for their suitability as the primary dye in the Gram stain. When a counterstain was not used, some dyes of all types were found to differentiate Gram-positive from Gram-negative organisms. When a counterstain was used, these dyes were found to vary greatly in their suitability. Those dyes found to be good substitutes for crystal violet were: Brilliant green, malachite green, basic fuchsin, ethyl violet, Hoffmann's violet, methyl violet B, and Victoria blue R. All are basic triphenylmethane dyes. Acid dyes were generally not suitable. Differences in the reaction of Gram-positive and Gram-negative cells to Gram staining without the use of iodine were observed and discussed but a practical differentiation could not be achieved in this manner. Certain broad aspects of the chemical mechanism of dyes in the gram stain are discussed.     

Use of the Gram stain in microbiology

The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be ''Gram-positive,'' whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be ''Gram-negative.'' This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as Gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.     

The characterization and ultrastructure of two new strains of Butyrivibrio

Strains B-385-1 and 2-33 are numerically important components rumen bacterial populations , but they have remained (taxonomically) undefined. In spite of some resemblance to Selenomonas ruminantium in their cell size and in their formation of tufts of flagella, they more closely resemble Butyrivibrio fibrisolvens in the subpolar location of their flagella, in their guanine + cytosine content, and in most biochemical characteristics, including butyrate formation. Cells of these strains stain Gram negative, as do both Selenomonas and Butyrivibrio, but their cell walls closely resemble those of Butyrivibrio in their Gram-positive type of molecular architecture and in their cleavage pattern in freeze-etching. Cells of these strains and of B. fibrisolvens have a very thin (ca. 12 nm) peptidoglycan cell wall; thus, they fail to retain the crystal violet complex of the Gram stain and stain Gram negative. This important structural characteristic of their cell walls places strains B-385-1 and 2-33 within the genus Butyrivibrio and certain morphological and biochemical characteristics distinguish them from B. fibrisolvens.     

Analysis of the Mechanism of Gram Differentiation by Use of a Filter-Paper Chromatographic Technique.

Bartholomew, J. W. (University of Southern California, Los Angeles), Thomas Cromwell, and Richard Gan. Analysis of the mechanism of Gram differentiation by use of a filter-paper chromatographic technique. J. Bacteriol. 90:766-777. 1965.-Data are presented which demonstrate that the mechanism of gram-positivity could not be due solely to factors such as a single, specific gram-positive substrate, specific affinities of crystal violet for certain cellular components, a specific crystal violet-iodine-substrate complex, or to any specific characteristic of the dye, iodine, or solvent molecules. Ruptured cells of gram-positive organisms stain gram-negatively when subjected to a standard Gram-stain procedure. However, when stained fragments of broken cells were deposited in thick layers on the surface of filter-paper strips and exposed to decolorizers, the rate of dye release correlated with the Gram characteristic of the intact cell. Therefore, the intact cell in itself is not an absolute requirement for Gram differentiation. The data are interpreted as indicating that the mechanism of Gram differentiation primarily involves the rate of permeation of molecules (dye, iodine, solvent) through the interstitial spaces of cell-wall material.     

Volutin as a Substrate of the Gram Reaction

A modified Gram procedure, with the use of an extremely diluted or acidified crystal violet solution, stained only volutin in contrast with nonstaining of the rest of cell in Gram-positive bacteria. The substrate of the Gram reaction is not only a ribonucleic acid-magnesium-protein complex in cytoplasm (Henry and Stacey 1946), but also a metaphosphate-ribonucleic acid complex in volutin and deoxyribonucleic acid in nuclei in Gram-positive cells. The isoelectric-point theory and permeability theory of the Gram stain are unsupported by the experiments.     

Effect of heat-staining procedure on the gram staining properties of mycobacteria]

Since the establishment of Gram stain by H.C.Y. Gram in 1884, it has been widely and routinely used as an aid for differentiation of bacteria. The bacteria are divided into three categories by the staining properties; Gram-positive, -negative, and -indefinite. All the text books in the world describe that mycobacteria such as M. tuberculosis are Gram-positive. By the merest chance, however, it was found that M. lepraemurium grown in tissues was not stained by the routinely used Gram staining method. Therefore, we tried to stain some of the mycobacteria by the Gram staining procedure which is widely used at present. The results obtained indicated that the mycobacteria tested were divided into three groups; the unstainable group such as M. leprae and M. lepraemurium, the Gram-positive and difficult-to-stain group which involves such slow growing mycobacteria as M. tuberculosis, M. avium, and M. intracellulare, and the Gram-indefinite group which contains such rapid growing mycobacteria as M. phlei, M. smegmatis, and M. chelonae. However, if Gram stain is carried out by the heating procedure at the first staining step, all the mycobacteria would become Gram-positive. Therefore, we emphasize that Gram staining of mycobacteria should be performed by the heating procedure.     

A simple metachromatic and fluorescent staining method for microorganisms using carbocyanine dye

The cationic carbocyanine dye, 1-ethyl-2-[3-(1-ethylnaphtho[1, 2d]-thiazolin-2-ylidene)-2-methylpropenyl]-naphtho[1, 2d]thiazolium bromide, interacts with several classes of anionic polymers, exhibiting metachromasia. We were able to stain various kinds of microorganisms with this dye. Gram-negative bacteria were stained reddish purple, while Gram-positive bacteria were stained violet or bluish purple. Stains of molds were of various colors. Yeast vegetative cells were stained reddish purple, but zygotic asci were bluish. Chlamydia trachomatis inclusions, which are surrounded by cytoplasmic membranes, were also stained red. Microorganism and cell stains have different features and can be identified also by use of fluorescent microscopy. The new staining method we report here is rapid and simple enough for routine microscopical examinations of smears of clinical specimens including microorganisms.     

Gram Staining Applied to Human Spermatozoa: a Simple Method for Studying Chromatin Condensation Status

Gram staining applied to human spermatozoa from fertile donors is described. The stain revealed populations of Gram-positive and Gram-negative spermatozoa. Data showed a significant and progressive decrease in the percentage of Gram-positive spermatozoa at different times during the chromatin decondensation procedure (SDS-BSA and SDS-EDTA). No significant correlation could be found between Gram staining and other functional tests used for spermatozoa; only the aniline blue staining test showed a poor correlation. Our study demonstrates that normal spermatozoa with regular chromatin condensation appear Gram-positive, while spermatozoa with altered chromatin condensation appear Gramnegative.     

Use of the Gram stain in microbiology

The Gram stain differentiates bacteria into two fundamental varieties of cells. Bacteria that retain the initial crystal violet stain (purple) are said to be 'Gram-positive,' whereas those that are decolorized and stain red with carbol fuchsin (or safranin) are said to be 'Gram-negative.' This staining response is based on the chemical and structural makeup of the cell walls of both varieties of bacteria. Gram-positives have a thick, relatively impermeable wall that resists decolorization and is composed of peptidoglycan and secondary polymers. Gram-negatives have a thin peptidoglycan layer plus an overlying lipid-protein bilayer known as the outer membrane, which can be disrupted by decolorization. Some bacteria have walls of intermediate structure and, although they are officially classified as Gram-positives because of their linage, they stain in a variable manner. One prokaryote domain, the Archaea, have such variability of wall structure that the Gram stain is not a useful differentiating tool.     

A Fluorescent Gram Stain for Flow Cytometry and Epifluorescence Microscopy

The fluorescent nucleic acid binding dyes hexidium iodide (HI) and SYTO 13 were used in combination as a Gram stain for unfixed organisms in suspension. HI penetrated gram-positive but not gram-negative organisms, whereas SYTO 13 penetrated both. When the dyes were used together, gram-negative organisms were rendered green fluorescent by SYTO 13; conversely, gram-positive organisms were rendered red-orange fluorescent by HI, which simultaneously quenched SYTO 13 green fluorescence. The technique correctly predicted the Gram status of 45 strains of clinically relevant organisms, including several known to be gram variable. In addition, representative strains of gram-positive anaerobic organisms, normally decolorized during the traditional Gram stain procedure, were classified correctly by this method.Gram’s staining method is considered fundamental in bacterial taxonomy. The outcome of the Gram reaction reflects major differences in the chemical composition and ultrastructure of bacterial cell walls. The Gram stain involves staining a heat-fixed smear of cells with a rosaniline dye such as crystal or methyl violet in the presence of iodine, with subsequent exposure to alcohol or acetone. Organisms that are decolorized by the alcohol or acetone are designated gram negative.Alternative Gram staining techniques have recently been proposed. Sizemore et al. (19) reported on the use of fluorescently labeled wheat germ agglutinin. This lectin binds specifically to N-acetylglucosamine in the peptidoglycan layer of gram-positive bacteria, whereas gram-negative organisms contain an outer membrane that prevents lectin binding. Although simpler and faster than the traditional Gram stain, this method requires heat fixation of organisms.Other Gram stain techniques suitable for live bacteria in suspension have been described. Allman et al. (1) demonstrated that rhodamine 123 (a lipophilic cationic dye) rendered gram-positive bacteria fluorescent, but its uptake by gram-negative organisms was poor. This reduced uptake by gram-negative bacteria was attributed to their outer membranes. The outer membrane can be made more permeable to lipophilic cations by exposure to the chelator EDTA (4). Shapiro (18) took advantage of this fact to form the basis of another Gram stain, one which involved comparing the uptake of a carbocyanine dye before and after permeabilizing organisms with EDTA. All of these methods, however, rely on one-color fluorescence, making analysis of mixed bacterial populations difficult.An alternative to the use of stains is the potassium hydroxide (KOH) test. The method categorizes organisms on the basis of differences in KOH solubility. After exposure to KOH, gram-negative bacteria are more easily disrupted than gram-positive organisms. This technique has been used to classify both aerobic and facultatively anaerobic bacteria, including gram-variable organisms (8). In a study by Halebian et al. (9), however, this technique incorrectly classified several anaerobic strains, giving rise to the recommendation that the method should only be used in conjunction with the traditional Gram stain.In this study we demonstrate a Gram staining technique for unfixed organisms in suspension, by using clinically relevant bacterial strains and organisms notorious for their gram variability. The method uses two fluorescent nucleic acid binding dyes, hexidium iodide (HI) and SYTO 13. Sales literature (11) published by the manufacturers of HI (Molecular Probes, Inc., Eugene, Oreg.), which displays a red fluorescence, suggests that the dye selectively stains gram-positive bacteria. SYTO 13 is one of a group of cell-permeating nucleic acid stains and fluoresces green (11). These dyes have been found to stain DNA and RNA in live or dead eukaryotic cells (16). Both dyes are excited at 490 nm, permitting their use in fluorescence instruments equipped with the most commonly available light sources. We reasoned that a combination of these two dyes applied to mixed bacterial populations would result in all bacteria being labeled, with differential labeling of gram-positive bacteria (HI and SYTO 13) and gram-negative bacteria (SYTO 13 only). The different fluorescence emission wavelengths of the two dyes would ensure differentiation of gram-positive from gram-negative bacteria by either epifluorescence microscopy or flow cytometry when equipped with the appropriate excitation and emission filters. While a commercial Gram stain kit produced by Molecular Probes includes HI and an alternative SYTO dye, SYTO 9, we are unaware of any peer-reviewed publications regarding either its use or its effectiveness with traditionally gram-variable organisms.     

Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay.

A well-known and widely used method for detection of siderophore production by microorganisms in solid medium is the universal chrome azurol S (CAS)-agar plate assay. However, the high toxicity of CAS-blue agar medium caused by the presence of a detergent impedes its utilization with many varieties of fungi and Gram-positive bacteria. To solve this problem, a modification of the CAS-agar plate assay was made by incorporating the CAS-blue dye in a medium with no contact with the microorganisms tested. Half of each plate used in our experiments was filled with the most appropriate culture medium for each type of microorganism and the other half with CAS-blue agar. This modification allowed us to study several strains of fungi (basidiomycetes, deuteromycetes, ascomycetes and zygomycetes) and bacteria (Gram positive and negative), some of them appearing for the first time in the literature. All the microorganisms grew properly and reacted in different manners to the CAS assay. Some strains of wood-decaying basidiomycetes (mainly white-rot fungi) and Aspergillus species produced the fastest color-change reactions in the CAS-blue agar. This modified method could facilitate optimization of culture conditions, since both CAS-blue agar and growth medium were prepared and added in the Petri plate separately.     

Protein secretion and surface display in Gram-positive bacteria

The cell wall peptidoglycan of Gram-positive bacteria functions as a surface organelle for the transport and assembly of proteins that interact with the environment, in particular, the tissues of an infected host. Signal peptide-bearing precursor proteins are secreted across the plasma membrane of Gram-positive bacteria. Some precursors carry C-terminal sorting signals with unique sequence motifs that are cleaved by sortase enzymes and linked to the cell wall peptidoglycan of vegetative forms or spores. The sorting signals of pilin precursors are cleaved by pilus-specific sortases, which generate covalent bonds between proteins leading to the assembly of fimbrial structures. Other precursors harbour surface (S)-layer homology domains (SLH), which fold into a three-pronged spindle structure and bind secondary cell wall polysaccharides, thereby associating with the surface of specific Gram-positive microbes. Type VII secretion is a non-canonical secretion pathway for WXG100 family proteins in mycobacteria. Gram-positive bacteria also secrete WXG100 proteins and carry unique genes that either contribute to discrete steps in secretion or represent distinctive substrates for protein transport reactions.     

Peptidoglycan remodeling and conversion of an inner membrane into an outer membrane during sporulation

Two hallmarks of the Firmicute phylum, which includes the Bacilli and Clostridia classes, are their ability to form endospores and their "Gram-positive" single-membraned, thick-cell-wall envelope structure. Acetonema longum is part of a lesser-known family (the Veillonellaceae) of Clostridia that form endospores but that are surprisingly "Gram negative," possessing both an inner and outer membrane and a thin cell wall. Here, we present macromolecular resolution, 3D electron cryotomographic images of vegetative, sporulating, and germinating A. longum cells showing that during the sporulation process, the inner membrane of the mother cell is inverted and transformed to become the outer membrane of the germinating cell. Peptidoglycan persists throughout, leading to a revised, "continuous" model of its role in the process. Coupled with genomic analyses, these results point to sporulation as a mechanism by which the bacterial outer membrane may have arisen and A. longum as a potential "missing link" between single- and double-membraned bacteria.     

Variations in the Gram Staining Results Caused by Air Moisture

The exposure of heat-fixed bacterial smears to relative humidities of 0, 52 and 98%, following the iodine step in a dry Gram stain procedure, markedly influenced the rate of decolorization upon exposure to 95% ethyl alcohol. If a single decolorization time were used to give a proper Gram differentiation after exposure to 98% relative humidity, this decolorization time might not result in proper Gram differentiation following exposure to 0% relative humidity. Different organisms varied in the degree of their response to changes in humidity. Hence the “degree of Gram-positivity,” as compared with other organisms, differed with changes in relative humidity. When Neisseria catarrhalis was compared with strongly Gram-positive and Gram-negative organisms, it was always found to be in an intermediate position in its Gram characteristics regardless of the relative humidity used. Because of the intermediate position of this organism, its proper Gram differentiation would require a precise definition of both the decolorization time and the decolorization procedure to be used. The results for all organisms studied clearly indicated that the control of wetness or dryness of the smear before decolorization would be essential in any Gram procedure where a single decolorization time is to be used.     

Differential Staining of Yeast for Purified Cell Walls, Broken Cells, And Whole Cells

Intact yeast cells are Gram positive but broken or disrupted cells are Gram negative. A counterstain with methyl green provides differential staining between cell wall and cytoplasm. The cells and cell fragments are dried on a slide and stained by a standard Gram stain. The preparation is then treated for 5 min with 1% phosphomolybdic acid, washed, and stained 0.5 min with 1% aqueous methyl green (unpurified by CHCl₃ extraction). Under these conditions whole, intact cells are dark purple or black, walls of broken cells and purified walls are light green, and the exposed cytoplasm stains light purple. All fractions can be easily differentiated.     

Variability of the Gram Reaction Report of Committee on Bacteriological Technic, of the Society of American Bacteriologists

The results of a cooperative investigation on the Gram stain are reported. One hundred and twenty slides were made by a single technician in one laboratory and distributed to ten collaborators. Each of these slides bore smears of six organisms, which were known to differ considerably from one another in their behavior to the Gram reaction. Identical directions were sent to all those taking part in the work as to how to perform the staining technic.

In regard to four of the six cultures fairly consistent reports were received from all those taking part in the tests. The other two cultures, however, proved so variable in their reaction toward the staining method that it is impossible to consider them either Gram-positive or Gram-negative. Such organisms must be regarded as belonging to an intermediate group, and should be called Gram-variable.

It is pointed out that these results agree with recent work, such as that of Churchman and of Steam and Steam; also that according to the theory of the latter investigators as to the relation between Gram reaction and the isolectric point of the bacteria, no sharp distinction between Gram-positive and Gram-negative organisms could be expected.

These considerations are very important when interpreting results of the Gram technic in the study of pure cultures; but they do not invalidate its use in diagnostic work where it is ordinarily employed to distinguish strongly positive from strongly negative organisms.     

Variability of the Gram Reaction Report of Committee on Bacteriological Technic,of the Society of American Bacteriologists

The results of a cooperative investigation on the Gram stain are reported. One hundred and twenty slides were made by a single technician in one laboratory and distributed to ten collaborators. Each of these slides bore smears of six organisms, which were known to differ considerably from one another in their behavior to the Gram reaction. Identical directions were sent to all those taking part in the work as to how to perform the staining technic.

In regard to four of the six cultures fairly consistent reports were received from all those taking part in the tests. The other two cultures, however, proved so variable in their reaction toward the staining method that it is impossible to consider them either Gram-positive or Gram-negative. Such organisms must be regarded as belonging to an intermediate group, and should be called Gram-variable.

It is pointed out that these results agree with recent work, such as that of Churchman and of Steam and Steam; also that according to the theory of the latter investigators as to the relation between Gram reaction and the isolectric point of the bacteria, no sharp distinction between Gram-positive and Gram-negative organisms could be expected.

These considerations are very important when interpreting results of the Gram technic in the study of pure cultures; but they do not invalidate its use in diagnostic work where it is ordinarily employed to distinguish strongly positive from strongly negative organisms.