Studies on the mechanism of the Gram stain

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A Thesis Presented to the Faculty of the Graduate School University of Southern California

In Partial Fulfillment of the Requirements for the Degree Master of Science In Bacteriology

fcy Tod Mittwer June 1950

UMI Number: EP55014

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T h is thesis, w ritte n by

Tod MItfewer under the guidance of h.%3... F a c u lty Gom mitteej and approved by a l l its members, has been presented to and accepted by the C o u n cil on G ra duate S tudy and Research in p a r t ia l f u l f i l l ­ ment o f the requirements f o r the degree of

Masfeer of Science in Bacteriology

F a c u lty C om m ittee

Chairm an


The author wishes to express appreciation of the encouragement of Dr. H. J. Conn, the invaluable advice of Dr. James W. Bartholomew, and of the financial aid given in the form of a research grant from the Biological Stain Commission.




I N T R O D U C T I O N ................................


REVIEW OF THE L I T E R A T U R E ...............


Permeability theories • • • • • • • • • • •


Chemical theories


............ . • • • •

Isoelectric point theory III.



MATERIALS AND M E T H O D S ...................... Source of organisms

.......... • • • •

10 10

Source of d y e s ..........


Testing dyes for suitability as primary s t a i n .................................... Primary stains


. • • • • • • • • • • • • •

Survey of iodine substitutes


. . . . . . .


Solubilities of dye-iodine precipitates in alcohol


.............. • • • • • •


Solubilities of precipitates in safranin solution

• .............

. . . . . . . .


Direct observation of iodine penetration into cells



• • • • • • • • • •

Effect of iodine on decolonization times


Quantitative determination of iodine penetration . IV.


R E S U L T S .....................................

15 18


PAGE Gram stain without counterstain Gram stain with counterstain

* ♦ . .

. • • . •


Survey of substitutes for iodine

. • •

Solubilities of dye-iodine precipitates

25 28

Solubilities of precipitates of dye plus iodine substitutes ...................


Staining of crushed cells • • • • • • • •


Visual observation of iodine penetration


Effect of iodine on decolonization time .


Quantitative determination of iodine penetration V. VI. BIBLIOGRAPHY

• • • « • «



37 42

. . . . ..........





PAGE The ability of various dyes to give differ­ entiation between Gram positive and Gram negative bacteria when used in a Gram staining procedure with and without a counterstain; and solubilities of dyeiodine precipitates


. . . . .


Iodine substitutes which gave Gram stains (using a counterstain)............



Iodine substitutes which did not give Gram stains (using a counterstain)




Solubility data for various precipitates of crystal violet plus iodine substitutes




Cell membrane penetration of Gram positive and Gram negative cells by iodine in aqueous and in alcoholic solutions . . .


Effect of iodine in alcohol on decolon­ ization time . . • • • • • * ...........




Quantitative estimation of aqueous and al­ coholic iodine penetration into Gram positive and Gram negative cells under standard conditions of time and concen­ tration



CHAPTER I INTROBUC TIGN Since Gram published his famous staining method (Gram, 1884); the Gram stain has been the subject of continu­ ous study and controversy.

The mechanism of this differen­

tial stain has been given particular attention.


theories of this mechanism are current, but no single ex­ planation is accepted generally, Gram*s original staining method was described as follows;

tissue sections or bacterial smears were treated /

with gentian violet for one to three minutes, and then placed in an aqueous solution of iodine and potassium iodide for one to three minutes.

The slide was then placed in

absolute alcohol until the tissue was completely decolorized, renewing the alcohol once or twice.

A counterstain of

Bismarck brown was then applied if desired.

Gram listed a

number of species of bacteria which retained the primary stain and a number of those which did not.

He also observed

that the nuclei of animal cells did not retain the gentian violet. This resume of Gramfs original description should clear up several misconceptions which appear in the litera­ ture.

It has been believed, for instance, that Gram did


not us© a counterstain, and it has also been said that Gram did not realise that some bacteria (now termed Gram negative bacteria) were decolorized during the alcohol step.

4 read­

ing of the original reference elucidates those points at once, and also indicates that Gram was conscious of the importance of his new staining method. Despite the large number of publications concerning this staining method, little is known regarding the specif­ icity of the reagents or procedures used.

Much of the

existing material in the literature is conflicting,


fore it was decided that any study of the Gram stain mechan­ ism must begin with such fundamental considerations as which dyes can be substituted for crystal violet, the reagents which can be substituted for iodine, the nature of the decolorizer, and the role of the counterstain. The large amount of interest shown in the Gram stain is due to its being the most important stain used in bacte­ riology today.

The Gram reaction is one of the most impor­

tant characteristics of a bacterial species both from a diagnostic and a classification standpoint.

It is also

recognized that the Gram staining reaction of a bacterial cell is closely related to deep seated physiological traits. An understanding of the mechanism by which this stain dif­ ferentiates between two groups of bacteria should aid in

the intelligent application of the Gram stain and in the elucidation of the fundamental differences between Gram positive and Gram negative bacteria. The literature bearing upon this problem will be reviewed briefly in the following chapter.

More detailed

references to specific points in the literature will be made in the discussion.

Detailed descriptions of all the

methods used in the experimental work are given in the chapter following the literature review.

Finally, the ex­

perimental results will be set forth, followed by a discus sion of their significance.


REVIEW OP THE LITEB4TURE The current theories of the mechanism of the Gram reaction can he grouped into three classifications, (1) cell membrane permeability theories, (2) chemical theories, and (3) the isoelectric point theory* Permeability theories*

Fischer (1903) made the

general statement that Gram positive bacteria were permeable and not plasmolyzable whereas Gram negative bacteria were impermeable and pl&smplyzable•

Brudny (1908) elaborated on

this concept, stating that the dye and iodine penetrated Gram positive cells and therein formed a dye-iodine pre­ cipitate with molecules too large to escape*

He believed

the Gram negative cells to be impermeable to iodine, thus preventing the formation of a precipitate.

Burke (1922) ,

however, indicated that the relative permeabilities were just the opposite.

By staining with an alkaline acetone

solution of a dye-iodine compound and then counter staining with safranin, the Gram reaction was reversed.

His ex­

planation was that the dye and iodine readily penetrated the Gram negative cells, staining them violet, whereas the dye and iodine (assumed by Burke to be present as a dyeiodine molecule) did not enter the Gram positive cells and


upon counter staining with safranin, the latter were red# Burke ascribed the permeability differences to the increased size of dye molecules when In combination with iodine, but St earn and S t e a m

(I960) showed photometrically that the

compound Is dissociated into its dye and iodine components in acetone solution# Barkers experiments modified but reinforced certain conclusions reached earlier by Benians (1912, 1920)• Benians1 observations indicated to him that the membrane of Gram positive cells was permeable to dye and Iodine, but impermeable to alcohol, thus preventing dissolution of the dye-iodine compound.

He then divided the Gram negative

bacteria into two types, (1) the Neisseria type with a dye, iodine, and alcohol permeable membrane permitting the dyeiodine precipitate to form inside, but also to be readily dissolved out by the decolorizer; and (2) the coli type with a dye-impermeable membrane preventing the entrance of the dye which is then merely adsorbed to the surface# Perhaps the most convincing evidence for the role of the cell membrane In the Gram stain was Benians* demonstra­ tion that crushed cells of any type always stained Gram negatively#

However, Schumacher (1926) claimed to have

disproved this, stating that cells crushed after the iodine step remained Gram positive#

It will be shown later that

Schumacherfs claim is not valid.


Kaplan and Kaplan (1933) presented additional evi­ dence for the permeability theory, and showed that the m e m ­ brane of Gram positive bacteria had a low permeability to iodine in alcoholic solution, whereas Gram negative cells were highly permeable to this iodine solution.

They showed

that the addition of iodine to the decolorizer displaced the equilibrium t© a degree which would also prevent the decolorization of Gram negative cells.

In addition Kaplan

and Kaplan confirmed a previous observation of Benians that the dye-iodine compound was much less soluble in alcohol than the dye alone, which resulted in additional slowing down of decolorization of Gram positive cells.

It is ob­

vious therefore, that while the permeability concept has had many adherents, there has been little agreement as to what substances were or were not permeable into the Gram positive and Gram negative cells. Chemical theories.

The first of the chemical theo­

ries was presented by XJnna (1S88) , who stated that the reaction of Gram positive cells was due to the formation of a triple compound of cell material, dye, and Iodine.


ever this concept was of very little use since Unna did not elaborate on the nature of the cellular substance concerned. Since that time much effort has been expended in an attempt to identify the specific cell material responsible.



workers (e.g., Tamura, 1914; Henry and Stacey, 1943) have claimed to have extracted substances which stained Gram positively independently of the cell, but no reports are found in the literature of anyone having duplicated these experiments. A number of workers have contended that the glycerides of the cell conferred Gram positivity.

As an example,

Dreyer, Scott, and Walker (1911) claimed that extraction with boiling ether rendered Staphylococcus Gram negative, and the extract appeared capable of rendering B. coll (Escherichia coli) Gram positive.

However such men as

Deussen (1920) and Schumacher (1926) have found that lipid solvents do not render cells Gram negative.

One also won­

ders if it is logical to conclude that glyceride removal would be the only effect of boiling ether on the chemical composition of the cells. Non-saturated lipids and lipoproteins have been cited by many as the essential Gram positive constituent. &mong others, Gutstein (1924) succeeded in rendering cells Gram negative by methods which were presumed specific for these substances.

He treated yeast cells with hot 2

then defatted with benzol and absolute alcohol.


He char­

acterized the Gram positive substance as an acidic lipid soluble in alcohol after hydrolysis, and stainable with basic dyes.

Other, and especially more recent work, has attri­ buted the Gram reaction to the nucleoproteins of the cell substance.

Beussen (1920) found that procedures which

destroyed nucleoproteins also caused cells to become Gram negative.

He was further able to restore Gram positivity

by appropriate treatments with nucleic acid.

Henry and

Stacey (1943) identified magnesium ribonucleate as a sub­ stance which could be removed and replated, depriving bacteria of their Gram positivity and restoring it, respect­ ively.

The work of Beussen and of Henry and Stacey has been

reproduced by others on many occasions and it appears quite obvious that magnesium ribonucleate is an essential com­ ponent of a Gram positive cell.

In addition Bartholomew

and Hmbreit (1944) rendered bacteria Gram negative with ribonuclease, the first time this conversion had been ac­ complished with a specific reagent. Beletang (1933), using carefully standardized tech­ niques, has shown that free lipids, nucleoproteins, and lipoproteins all contribute to Gram positivity.


these three substances in succession, he found that cells became progressively less Gram positive as measured by the time required to decolorize. Isoelectric point theory.

Stearn and S t e a m (1924)

proposed that Gram positive organisms have a lower iso-


electric point than Gram negative organisms, due to their higher content of more acidic substances.

According to this

concept, iodine oxidizes eellular material, particularly unsaturated lipids, making these substances more acidic* This effect is greater on Gram positive cells than on Gram negative cells and separates their isoelectric points even further.

The more acidic Gram positive cells will there-

fore have greater affinity for basic dyes and will resist decolorization longer.

A considerable amount of evidence

has accumulated to weaken this theory greatly and this will be discussed in greater detail later.


MATERIALS AMD METHODS Wherever possible, an attempt was made to use care­ fully standardized, reproducible techniques in order that personal errors would be reduced to a minimum* Source of organisms *

All cultures used were obtained

from the stock culture collection of the Department of Bacteriology, University of Southern California*


otherwise specified, all tests were made with eighteen hour, nutrient agar slant cultures, grown at 37° G. Source of dyes*

Many of the dyes used were obtained

from Dr. H. J. Conn, Biological Stain Commission, Geneva, New York.

Others were obtained from National Aniline Com­

pany, New York, N* Y . , and some were from the Department of Bacteriology, University of Southern California.

The dyes

are identified in the tables of results by their certifica­ tion numbers or lot numbers wherever these were known. Testing dyes for suitability as primary stain* were made up in 0*5$ solution as follows %


0*25 gram dye

was dissolved in 1 ml. of 95$ ethyl alcohol and diluted to 50 ml. with distilled water. near neutrality.

The pH was then adjusted to

A n effort was made to include at least


on© representative of each type of dye used in bacteriologi­ cal staining# The test organisms were Bacillus subtilis, Neisseria catarrhalis, and Escherichia coli. grown on nutrient agar slants at 37° C. for 18 hours.

The cells were suspended in

distilled water, the turbidity adjusted to approximately that of a No. 8 barium sulfate standard, and a loopful (calibrated to contain 0.01 ml.) spread over approximately one square centimeter on the slides. thin unifoim smears.

This procedure gave

One smear of each of the three organ­

isms was placed on a slide.

A sufficient number of slides

were prepared to last through most of the experiments. Two modifications of the Gram stain were useds


the Hucker-Conn (1923) method, following the procedure and using reagents as given in Diagnostic Procedures and Reagents (A.P.H.&., 1945), but emitting the ammonium oxalate. This technique involves staining at about pH 6.4; (2) the Kopeloff-Beerman (1922) method.

In this modification

NaHGO-j is added, raising the pH of the primary stain to about 9.3.

The iodine solution is also alkaline.

Safranin or methylene blue in 0.25$ solutions were used as counterstains, depending upon the color of the dye used as primary stain. The dyes were rated on an arbitrary scale of 1 to 10 as to their ability to differentiate Gram positive from


Gram negative cells, the degree of differentiation given by crystal violet being taken as standard and assigned, a rating of 10 ♦ Primary stains*

Smears were stained with 0*5$ solu­

tions of the various dyes for one minute*

The dyes were

then rated by three observers on a scale of 1 to 10 as to their intensity and depth of color as a primary stain*


intensity of a crystal violet stain made in this manner was arbitrarily taken as standard with a value of 10* Survey of iodine substitutes*

The Hucker-Gonn (1923)

modification was used for Gram staining exactly as recom­ mended except that other reagents were substituted for the iodine solution*

Test organisms were Saccharomyces cere-

visiae. Bacillus subtilis t Neisseria catarrhalis. and Escherichia coli, grown on nutrient agar slants at 37° C* for 18 hours* prepared*

4 large number of identical thin smears were

Four stains were made at different times before

discarding any reagent as giving negative results*

411 those

giving good results were tested at least twelve times.


giving variable results were tested about twenty times. Concentrations of iodine substitutes used were 1$, unless saturated solutions were less than this amount.


ally a reagent in 1$ concentration bleached the dye; in this


case the reagent was diluted until bleaching no longer oc­ curred. Solubilities of dye-iodine precipitates in alcohol. The method of Holmes (1927) , slighly modified, was used to determine the solubilities in alcohol of the precipitates. Gramfs iodine was added to an aqueous 0.5$ solution of the dye until the dye was precipitated.

The precipitate was

centrifuged out and washed several times with water.


excess of this precipitate was added to 95$ alcohol in large tubes.

The tubes were sealed and agitated intermittently in

a shaking device for five days at 26° G.

The saturated

solution was then filtered off, a measured volume placed in a weighing dish and evaporated.

The residue was dried to

constant weight at 60° C. and the weight obtained by dif­ ference.

The solubility was then calculated as grams per

100 ml. of solution. Solubilities of precipitates in safranin solution. Accurately prepared aqueous solutions of dyes of 0.25$ con­ centration were made.

These were diluted to concentrations

ranging from 0.0001$ to 0.0015$ and, using appropriate filters, standard curves were constructed from KlettSummerson photoelectric colorimeter readings.

These curves

had the concentration of dye on the abscissa and the Klett


readings on the ordinate and were used to determine concen­ trations from subsequently made Klett readings.

The filters

used were selected from those available having the nearest transmission band to the absorption maximum of the dye being determined, as follows: Absorption Maximum, m u

Bye Crystal violet Malachite green Ethyl violet Hoffmann*s violet Methyl violet B Victoria blue R Brilliant green Victoria blue B Toluidine blue Rile blue sulfate Methylene blue Thionin Methyl green Jkzure A Azure B Celestin blue B

591 616 596 596 (?) 583 615 623 619 635 644 667 602 634 638 652 654

Filter Ho. used 59 62 59 59 59 62 62 62 62 62 66 59 62 62 66 66

To determine the solubility of dye-iodine precipi­ tates in safranin, e.g., to determine the concentration of one dye in the presence of another, the following method was used:

precipitates as previously prepared were added

in excess to 0.25$ safranin, and agitated at 26° C. for twenty-four hours and then filtered.

The filtrate was

diluted to various concentrations and read in the Klett apparatus, using identical dilutions of safranin alone as



'The proper filter was used so that the concentra­

tion of the measured dye could be determined in the presence of the safranin.

These readings were translated into per­

centage concentrations on the empirical curves previously prepared. well.

Three trials were made and the results agreed

The data given are averages of these trials. Solubilities of precipitates of crystal violet plus

various substitutes for iodine were determined in the same manner. Direct observation of iodine penetration into cells. A wet mount technique was used to observe staining with iodine solutions on heat fixed but otherwise unstained and untreated cells.

Penetration was indicated by visual obser­

vation of staining of the protoplasm of the cells. Bffect of iodine on decolonization times.


stains were made on a large number of slides, and decolor­ ization was performed by immersing the slides in absolute alcohol containing 0.33% iodine, and in absolute alcohol alone.

Slides were withdrawn at intervals and counter­


The smears were then observed microscopically and

the color of the cells determined. Quantitative determination of iodine penetration. One loop (calibrated to contain 0.01 ml.) of cells was added


to two milliliters of 0*33$ iodine in 1$ aqueous potassium iodide solution, and one loop of cells was added to two milliliters of 0*33$ iodine in 95$ alcohol.

The cells were

suspended by shaking for two minutes, and were centrifuged for two minutes at 6,000 r.p.m.

The supernatant was poured

off and the cells were re suspended in three milliliters of 95$ alcohol.

This step washes off iodine adsorbed to the

surface of Gram positive cells and extracts a major portion of the iodine from the Gram negative cells.

The Gells were

centrifuged again for two minutes and the supernatant dis­ carded.

Three milliliters of 1$ aqueous potassium iodide

solution were added to extract the iodine and the cells were suspended and shaken for one minute.

The cells were then

centrifuged for three minutes and the supernatant containing the extracted iodine was poured into a Klett tube.


milliliters of 0.15$ soluble potato starch solution were added, the tube shaken and read immediately in the Klett apparatus, using the green (540 uy