J. W. Bartholomew

Variables Influencing Results, and the Precise Definition of Steps in Gram Staining as a Means of Standardizing the Results Obtained

Results of a Gram staining procedure varied with modifications of each of the steps involved. The best Gram differentiation was obtained when crystal violet and iodine solutions of high concentrations were used, and when n-propyl alcohol was used as the decolorizer. The decolorization step must be carefully quantitated, and one of the most important variables observed was whether a slide was brought into the decolorizer wet, or dry. Dry slides took 6 to 12 times as long to decolorize as wet. Wash steps, following crystal violet, and following the decolorizer, also greatly influence results by causing Gram-positive organisms to appear to be Gram-negative. The results indicated that Gram-stain procedures should not be varied to suit the whims of individual operators, and that each step could be specifically defined both as to the reagent used, and the procedure to be followed.The followng Gram procedure is recommended for heat-fixed bacterial smears on glass slides. Flood the slide with Huckers crystal vio...

A Simplified Bacterial Spore Stain

A simplification of the Schaeffer-Fulton spore stain for bacteria is presented. It is shown that omission of the heating step during staining with malachite green resulted in spore stains as good as when the heat was applied. The simplified procedure involves (1) heat fixation of the smear by 20 passages through the flame, (2) staining with saturated aqueous malachite green for 10 minutes, (3) rinsing, and (4) counterstaining with 0.25% aqueous safranin for 15 seconds. The omission of the heating step in staining has obvious advantages, particularly in the classroom.

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 aque...

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, Hoffmanns 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.

DYE EXCHANGE IN BACTERIAL CELLS, AND THE THEORY OF STAINING

Bacterial cells were stained in sequence, and at various pHs, by 22 different basic dyes. It was found that any dye could replace another already present in the bacterial cell. This replacement was shown to act according to mass action laws for reversible reactions, and hence was influenced by concentration of reagent and time of application. Since basic dyes are also known to react at carboxyl group sites, the phenomena of staining of bacterial cells by ordinary basic dyes must be a chemical adsorption exchange reaction.

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 c...

Electron microscope studies of nuclear changes in Saccharomyces cerevisiae during bud formation.

Ultraviolet photolysis was used to obtain transparent yeast cells when observed with the electron microscope. Following proper irradiation the yeast nucleus was easily discernable and appeared to be in various stages of division. Spindle-like figures were seen which give weight to the concept of a mitotic process for the division of the yeast nucleus. Distinct chromosomes were not observed.

Quantitative Determination of Dye Uptake by Bacterial Cells

A colorimetric method is presented for the quantitative determination of dye uptake by bacterial cells. Experiments showed that the dye to cell ratio was of major importance in controlling the amount of dye taken up per weight of bacterial cells. Approximate dye saturation of cells could be obtained (at pH 6.1 to 6.3) although the dye uptake curves did not absolutely level off.

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 de...

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 ...