Studies in Gram Staining

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuchsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal viokt-stained organisms with alcoholic safranin (0.25%) for 15 scc will distinguish Gram-positive bacteria (viokt) from Gram-negative bacteria (pink).

Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily dccolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.     

Studies in gram staining.

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuschsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal violet-stained organisms with alcoholic safranin (0.25%) for 15 sec will distinguish Gram-positive bacteria (violet) from Gram-negative bacteria (pink). Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily decolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.     

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.     

A Differential Stain Favorable to the Diagnosis of Neisserian Infection

A differential Gram stain has been evolved which incorporates the combined features of the original Gram and Pappenheim methods. National Aniline crystal violet and new methyl green and pyronin are the dyes preferred. The iodine mixture of Kopeloff and Beerman is a satisfactory mordant and Merck's pure technical acetone is an excellent differentiating agent. A system is established by means of the dyes and reagents which form a physicochemical equilibrium, provided pure dyes are employed, and the technic is carried out with precision. Gram-positive bacteria are coated by means of buffered crystal violet solution and the iodine-sodium hydroxide solution precipitates the crystal violet from other substances. The dye-iodine precipitate is readily dissolved by pure acetone. Iodine green, a pure derivative of crystal violet has the effect of noninterference in the technic and has selective action upon nuclear substance. Pyronin has affinity for Neisserian organisms primarily and acts as an inert substance upon most other proteins, (except cytoplasm of eosinophils, lymphocytes, plasma cells, and endothelial cells). The following technic is recommended:

Stain air-dry films 3 to 5 minutes in a 1% solution of crystal violet in 10 parts of Clark and Lubs' phosphate buffer of pH 6.6 to 7.0 and 90 parts water. Decant and flush with 2% iodine in N/10 NaOH. Decant and decolorize in acetone 10 seconds or less. Air dry and counterstain 1 1/2 to 2 minutes with methyl-green-pyronin (2 parts 2% aqueous methyl green National with one part 0.3% aqueous pyronin yellowish). Wash and air dry. Oil of Bergamot is preferable to xylene as a clearing agent. Best results are obtained if each slide is handled separately as for staining blood films.     

Some Staining Methods for Bacteria and Yeasts

For staining flagella of bacteria use actively motile organisms 20 to 24 hours old, allow to diffuse in sterile water 20 to 30 minutes, transfer droplets of the suspension to clean slides and let evaporate without spreading. Then treat 2 to 4 minutes with the following mordant: tannic acid 10 or 20%, 50 cc.; ferric chloride 5%, 10 to 15 cc.; carbol fuchsin (Ziehl-Nielson), 5 cc.; hydrogen peroxide 3%, 6 to 8 cc. Wash and stain 2 to 3 minutes with a mixture of basic fuchsin, saturated alcoholic, 10 cc.; anilin oil (1 part) and 95% alcohol (3 parts) mixed, 5 cc.; distilled water, 30 cc.; acetic acid, 4%, 1 cc. Wash thoroly with water.     

The Germicidal Effect of Staining Solutions

Gentian violet, crystal violet and carbol fuchsin applied to cover slip preparations for one minute will destroy the majority of non-spore-forming bacteria and yeasts, tho they can not be relied upon to do this consistently and in all cases.

The Gram staining procedure is more effective and non-spore-formers were never found to survive this process.

Methylene blue stains exert very little if any germicidal power and most organisms survived them readily. India ink was totally ineffective.

Several species of yeasts and yeast-like molds were killed in every instance by the Gram stain, gentian violet, crystal violet and carbol fuchsin, but survived both Loeffler's methylene blue and a plain aqueous solution of methylene blue.     

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.     

Feulgen's Reaction Applied to Protozoa and Small Worms, Mounted In Toto in Venetian Turpentine

Permanent mounts of certain protozoa and small worms are obtained as follows: kill suspensions of the organisms with Feulgen's fixative (6% HgCl₂ in 2% aqu. acetic acid) for 3 to 24 hours. After pipetting off the fixative, add successively: 70% iodized alcohol; ditto, 30 minutes later; 50%, then 35% alcohol; 2 baths distilled water; normal HCl. Transfer to cold water and heat to 60°C for 4 to 5 minutes or longer. Cool under running water; and wash in distilled water.

Stain 1 to 3 hours in Feulgen's fuchsin sulfurous acid (1 g. of a suitable basic fuchsin, e. g. rosanilin hydrochloride, boiled in 200 cc. water, cooled, and allowed to stand 24 hours after adding 20 cc. normal HCl and 1 g. sodium bisulfite). Pass thru 3 baths of 200 cc. distilled water with 10 cc. normal HCl and 1 g. sodium bisulfite. Transfer to water and then to 35%, 70%, and 95% alcohols successively. Counterstain with fast green FCF, orange G or eosin Y in 95% alcohol. Pass thru two changes of absolute alcohol.

Transfer to 10% Venetian turpentine and place in a dessicator; mount after the turpentine has become concentrated.

If sections instead of total mounts are desired, the material should go from absolute alcohol, thru alcohol-xylol and xylol to paraffin (or preferably paraffin of M. P. 56°C with 3% bees-wax). The paraffin may be added to the material in the test tube, and cooled after the organisms have settled. Then break the tube, trim a block, and cut.     

Metallic Lakes of the oxazines (Gallamin Blue,Gallocyanin and Coelestin Blue) as Nuclear Stain Substitutes for Hematoxylin

Becher's investigations upon the soluble metallic lakes of the oxazines have been re-investigated, extended and results described. Gallamin blue, gallocyanin and coelestin blue in combination with ferric ammonium sulfate gave the best results. The dyes are dissolved in a five per cent aqueous solution of ferric ammonium sulfate. The solution is boiled for 2–3 minutes, cooled, filtered and ready for immediate use. The iron lakes of these dyes stain nuclei excellently giving a deep blue or blue black in 3–5 minutes. No differentiation with acid is required. Coelestin blue gives the most stable solution and is recommended as a routine nuclear stain. The protoplasm remains practically colorless and counter-staining with acid dyes such as ethyl-eosin, orange G, or fuchsin gives pictures which cannot be distinguished from a good hematoxylin stain.

Counter-staining with van Gieson solution is also possible. Benda's modification of the van Gieson solution is recommended. Staining of fat with Sudan, scarlet red, etc., does not interfere with nuclear staining by these dyes.

As applied to the central nervous system these dyes are far superior to hematoxylin. Ganglion and glia cells are as excellently stained as with thionin.

The most widely used fixatives, namely formaldehyde, Mueller-formaldehyde, Zenker's and alcohol, give equally as good results. The nature of the staining process is briefly discussed and a prospectus offered.     

Delafield's Hematoxylin and Safranin for Staining Meristematic Tissues

The following technic is suggested for staining permanent preparations of meristematic tissues: Prepare and mount the sections by the usual paraffin method. From water, stain them 2-10 minutes in a solution made by adding 2-4 cc. of Delafield's hematoxylin to a Coplin jar full of tap water. As staining is progressive, the sections should be examined from time to time with a microscope. When the cell walls have become a deep purple, transfer the preparations, thru the usual series, to a mixture of xylol-absolute-alcohol in equal parts, and from this to a counterstain made by adding 4-6 cc. of a saturated solution of safranin in absolute alcohol to a Coplin jar full of xylol (75%) with absolute alcohol (25%). This stains the nuclei. Leave the sections in the counterstain at least 2 hours and then rinse them in xylol-absolute-alcohol (1:1) to remove excess safranin. Transfer them to pure xylol and then mount them in neutral balsam.     

Gram Reaction in Crushed Yeasts

Thick smears of yeasts are dried, crushed between slides or cover-slips, stained with anilin crystal violet 11/2 minute, Lugol's iodine solution 2 minutes, decolorized in undiluted anilin oil, and counterstained with 1% aqueous safranin. Photomicrographs are given of crushed yeasts, thus stained, which show a Gram-positive outer layer and a Gram-negative inner substance.     

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 Hucker's crystal violet for 1 ruin. Wash for 5 sec by dipping into tap water running into a 250 ml beaker at a rate of 30 ml per sec Rinse off the excess water with Burke's iodine, flood the slide with this solution for 1 min, then wash 5 sec in tap water as above. Decolorize by passing the wet slide through 3 (75 × 25 mm) Coplin dishes containing n-propyl alcohol, decolorize 1 min in each dish for a total of 3 min. Wash 5 sec in tap water as above, rinse off the excess water with 0.25% safranin, then flood the slide with this solution for 1 min. Wash as above, blot dry, and examine. An alternate procedure for decolorization would be to use either 95% n-propyl alcohol or 95% ethyl alcohol, but shorten the decolorization time to 30 sec per dish for a total of 1.5 min. After 10 slides, the decolorizer in the first dish should be replaced by fresh. This dish is then placed last in the sequence, with dish No. 2 moved to the No. 1 position.     

A Technic for Differential Staining of Nucleoli and Chromosomes

A procedure is described which enables a stain to be definitely located in the substance of the nucleolus. Material is fixed in either Navashin or Levitsky; the chromatin is stained by means of the improved Feulgen technic introduced by de Tomasi, and preparations brought thru the washing solutions down to distilled water. From distilled water the material is transferred to a mordant solution, 5% sodium carbonate in water, in which it is left for at least one hour. After mordanting wash well with water then stain for ten minutes in light green solution (90% alcohol, 100 cc, light green SFY, 0.5 g, aniline oil, 2 drops, well shaken); differentiate in alcoholic sodium carbonate solution, (70% alcohol saturated with carbonate); treat with 95% alcohol, absolute alcohol, equal parts xylene and absolute alcohol, clear in pure dry xylene and mount in neutral balsam. Cytoplasm and karyolymph should be quite clear, with magenta chromatin and well defined green nucleoli. The light green does not behave like a simple counterstain as in previous technics but as a definite stain for nucleolar material.     

Cytological Methods for Crepis Species

Root tips of Crepis species are fixed in La Cour's “2BE” and dehydrated thru a butyl alcohol series. They are stained in 1% crystal violet for 1 hour, with chromic acid and iodine as pre-and post-staining mordants, respectively, and passed thru dehydrating alcohols containing picric acid and ammonium hydroxide. Differentiation is done in clove oil. The method is rapid; the chromosomes are dark purple; the centromere is not stained; and the cytoplasm is clear. By further controlled destaining the hetero-chromatic segments within the chromosomes may be located.

Pollen mother cells are fixed in acetic alcohol (1:4) and squashed in aceto-carmine. A method is described for making semi-permanent preparations mounted in diaphane.

Pollen grains are mounted in lacto-phenol with acid fuchsin or anilin blue W. S. as the dye.     

A Note on the Gram Stain

A modification of the Gram stain in which iodine-alcohol is substituted for 95% alcohol as a decolorizing agent has been found particularly useful in staining Gram-positive organisms in tissues and also for smears. The technic for tissue sections follows:

1. Apply nuclear stain.
2. Wash.
3. Stain in Hucker's gentian violet 2 to 3 minutes (i. e. 1 part Sat. Alc. Sol. crystal violet to 4 parts 1% Aqu. Sol. ammonium oxalte).
4. Wash in water.
5. Stain in Gram's iodine 5 minutes.
6. Wash in water.
7. Decolorize in 95% alcohol to which enough tincture of iodine has been added to give a mahogany color.
8. Counterstain.
9. Dehydrate and mount.

     

Stain Technic for Nissl's Granules Following Alcohol-Formol Fixation

1. Nerve tissue is fixed 2-4 hrs in a 5% solution of strong formalin in commerical 95% alcohol.

2. If dehydration is perfect, either chloroform or xylol may be used as a clearing agent.

3. A slow method of paraffin infiltration is advisable.

4. Sections should be cut 10-12 microns in thickness.

5. Coplin staining jars should be annealed by placing them on a rack in a pan of cold water, bringing the water to the boiling point, and allowing the jars to stand in boiling water for twenty minutes.

6. One per cent aqueous solutions of either methylene blue or Grübler's Neutral Roth are used as specific stains for Nissl's granules.

7. These stains are heated to boiling in a beaker, the slides are placed in the Coplin jars which are partially submerged in boiling water, and the hot stain poured into the jars. The flame beneath the water bath is turned down and the slides left for 20 minutes.

8. The excess of primary stain is washed off in 25% and 50% alcohol and the slides passed rapidly thru the alcohol series to absolute alcohol, and finally to xylol.

9. When counterstaining is desired, nigrosin in 1% aqueous solution, methyl orange, saturated solution in 50% alcohol, or, a 0.5% solution of eosin in 50% alcohol are recommended. These stains are used cold, and the slides are merely dipped in them after the excess of primary stain has been washed out in 25% and 50% alcohol.

10. If a cold primary stain is desired, a saturated solution of thionin in distilled water, acidified with 1% carbolic acid, will prove specific for the Nissl substance. Sections should be stained 5–10 minutes in thionin, then passed rapidly thru to absolute alcohol, and xylol. The same counterstains may be used as in the hot method.

11. Sections prepared by the hot method show little tendency to fade after ten years use.

12. Excepting neutral red, all the stains used in this technic are carried by the National Aniline and Chemical Company and are satisfactory. Coleman and Bell neutral red may be substituted for Grübler's Neutral Roth with good results.     

Detailed Differentiation of Bacteria by Means of A Mixture of Acid and Basic Dyes at Different pH-Values

As previously reported by the author (1927), a mixture of methylene blue and eosin Y can be used for the differential staining of bacteria. It gives a fairly deep staining of bacteria at about pH 3 and above. Below pH 3 the eosin Y stains bacteria only a very pale pink; at such high H-ion concentration, the eosin is present as undissociated color acid, and for this reason not enough eosin is in solution to stain bacteria. To improve the staining at such reactions, the eosin was replaced by a stronger acid dye, namely acid fuchsin. The mixture of methylene blue medicinal Merck and acid fuchsin can be successfully used at a pH-value as low as 0.8. The method of staining by this new mixture is entirely the same as with the old mixture. It is sensitive enough to detect the difference in the isoelectric points: (1) of the single bacteria from the same pure culture, (2) of different strains of the colon and typhoid organisms. Some strains of the colon organism were found by this method with an isoelectric point at a pH-value as low as that of the Staphylococcus. Others, on the contrary, have their isoelectric point as high in the pH-scale as that of the typhoid organism. The new mixture can also be used for the study of the chemical composition of the different parts of bacterial body. Applying it at a definite pH-value, the author was able to stain differentially polar bodies of the typhoid group and of the diphtheria organism. This new mixture can be recommended in staining of B. diphtheriae as a substitute for Neisser's stain. It is interesting to note that polar bodies of the colon group consist of more alkaline protein than the body of the bacteria itself, i. e., they are stained by acid fuchsin. The polar bodies of the B. diphtheriae on the contrary are composed of more acid protein than the bacterial body; i. e., they are stained by methylene blue. The impossibility of detecting the above mentioned variations in the isoelectric points of bacteria using the Gram method is explained by the absence of pH variations in the latter technic. The differentiation of bacteria by the Gram stain depends chiefly on the varying stability of the compound formed (Gram-positive or Gram-negative bacteria plus gentian violet and iodine) in the presence of organic solvents, such as alcohol, acetone, etc.     

The Smear Technic in Plant Cytology

The following method of making permanent smears of pollen mother cells is in general use and gives excellent results. Determine the stage of meiosis from aceto-carmin mounts. Smear the pollen mother cells on a dry slide. Fix in Navaschin's or a modified Flemming's solution from 1 to 2 hours. Wash in 10 to 20% alcohol from 15 to 30 minutes. Stain in 1% aqueous crystal violet from 1 to 5 minutes. Rinse in water and pass thru 30 to 50% alcohol, about 15 to 20 seconds in each. Transfer to 80% alcohol containing 1% iodine and 1% potassium iodide for 30 seconds. Destain with absolute alcohol, followed by clove oil. xylol, balsam and cover.

Permanent smears for chromosome counts can be quickly made by smearing pollen mother cells on a dry slide, fix and stain with aceto-carmin, dehydrate with mixtures of absolute alcohol and acetic acid, follow with xylol, balsam, and cover.     

A Safe Spore Stain for Class Use

In looking for an efficient method of pre-treating bacterial spores for staining with carbol fuchsin, the author found that the ammonia in Lagerberg's formula and the chromic acid in Moeller's formula were both fairly effective in increasing permeability. Combining the two proved highly successful; and the following method has been used for four years by classes of beginners in the study of spore-formers isolated from soil by the students.

Cover the heat-fixed smear with five percent chromic acid and after thirty seconds add twice the amount of concentrated ammonia to the acid. After two minutes rinse and steam with carbol fuchsin for two or three minutes. Rinse, destain with one percent sulphuric acid for fifteen to thirty seconds, rinse again and flood slide with tap water. Add to this a few drops of Loeffler's methylene blue and allow to counter-stain for ten to thirty seconds. Rinse, blot, dry and examine. If instructions are followed the results are always good.     

Gram staining and lectin binding properties of Myxosporea and Sporozoea

The staining method developed by Christian Gram was introduced as a simple and highly selective tool for demonstrating myxosporean and coccidian sporogonic stages. When using standard blood staining procedures for those enigmatic parasites it is sometimes difficult to distinguish them from fish host tissue. They clearly exhibit a partial Gram-positive reaction in histological sections, but staining is variable in air dried fish organ imprints. To visualize the Gram-negative background of different host tissue components in histological sections, the conventional safranin counterstain of the Gram protocol may be modified as follows: after application of 2% crystal violet (basic violet 3) and Lugol's solution, sections are stained with 0.1% nuclear fast red-5% aluminum sulfate and 0.35% aniline blue (acid blue 22) dissolved in saturated aqueous picric acid. Replacement of the Gram-specific dye crystal violet with 2% malachite green gave similar results in organ imprints containing myxospores or coccidia, but only in sections containing myxosporea. Staining for 1 min with an aqueous solution of 0.5% malachite green and followed 1 min washing was sufficient for rapidly demonstrating the parasite spores in organ imprints of both myxosores and oocysts. With regard to the role of acid mucopolysaccharides and other carbohydrates in the Gram reaction of spores, alcian blue 8GX staining was compared to the binding of FITC-labeled WGA, GS I and GS II. Each lectin was applied at 20 μl/ml PBS, HEPES for 1 hr. Whereas WGA yielded a nonspecific pattern like the alcian blue staining, GS II resulted in a pattern similar to the Gram staining results. This binding was weak in untreated specimens, but was significantly enhanced when digested first within trypsin overnight in a humid chamber at 37 °C. The binding of GS II to both myxosporidian and coccidian spores suggests that they are both composed of polymers containing N-acetyl-D-glucosamine residues. Furthermore, the results suggest that this hexosamine plays a key role in the Gram reaction.