THE GRAM STAIN AND DIFFERENTIAL STAINING

M. L. KAPLAN AND LEAH KAPLAN

Bureau of Laboratories, Department of Health, New York City
Received for publication, June 22, 1932
The Gram test is the most commonly employed method for
differential staining of bacteria. Despite this fact, and the con-
siderable time which has elapsed since its discovery, its mechanism
is clouded in doubt. The r6le of iodine in the test and the differ-
ence between positives and negatives are still controversial mat-
ters. It seems to be generally known that the triphenylmethane
dyes form water-insoluble compounds with iodine; but Stearn
and Stearn (1930) attribute the fixation of the dye in Gram-posi-
tives to an oxidation of the cell material, rather than to the forma-
tion of this compound. Burke and Barnes (1929), on the other
hand, ascribe the retention of the dye to the increased size of the
dye-iodine molecule, and postulate that the essential difference
between positives and negatives resides in the lower permeability

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of the cells of the former type to the compound. This conception
is negatived by the observation of Stearn and Stearn that the dye-
iodine compound dissociates into its components in alcohol solu-
tion. Among other views of the Gram mechanism are those of
Brudny (1908) who considers that Gram-negatives are imper-
meable to iodine; and of Benians (1912), who suggests a lack of
permeability of positives to alcohol.
Benians' concept of a difference between positives and negatives
in alcohol permeability would explain the so-called "pseudo-
Gram" reaction obtained by the use of Victoria Blue and other
alcohol-soluble dyes, such as Spirit Blue and Night Blue, if we
assume that the dissolved dye molecule unites with several
molecules of the solvent. This would involve the passage of a
considerable number of solvent molecules into and out of the
stained cell. However, since alcohol does extract methyl and
309
310 M. L. KAPLAN AND LEAH KAPLAN

crystal violet from all bacteria, the positivity resulting from iodine
treatment still requires explanation. In order to test the solubil-
ity of the crystal violet-iodine complex in alcohol, equimolar solu-
tions of the two were mixed and the concentration observed at
which a noticeable precipitate was formed. This occurred at a
concentration of 0.5 per cent crystal violet.' This is about one-
tenth the solubility given for gentian violet, a dye mixture com-
prising methyl and crystal violet. The low solubility of the
compound is not incompatible with the observations of Stearn
and Stearn previously cited, as to the dissociated state of the dye-
iodine compound dissolved in alcohol, and is probably due to an
equilibrium between the associated and dissociated states, the
former being only slightly soluble. The fact that precipitation
leaves a violet supernatant solution, while Gram-stained bacteria
are usually blue, can be considered as confirmation of the observa-
tions of Stearn and Stearn.
If we consider that the removal of a dye molecule from the
interior of a cell requires a driving force supplied by osmotic pres-
sure, and hence proportional to concentration, the reduction of its
solubility to one tenth undoubtedly retards extraction. The
Gram test, which really amounts to the slowing down of dye ex-

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traction by the alcohol, may then be explained as a low permeabil-
ity of positives to the dye. But with the entrance of alcohol into
the cell a solution of iodine is formed as well as one of dye. Con-
sequently, the removal of the dye can only take place if it is ac-
companied by an escape of iodine; since otherwise the iodine
remaining behind would precipitate the dissolved dye in accord-
ance with the mass law. In other words, permeability to both dye
and iodine is necessary if decolorization is to take place. While it
is true that aqueous Gram iodine solution penetrates Gram-posi-
tives as well as negatives (which we have proved by the rapid de-
colorization of methylene blue stained bacteria by Gram iodine),
it must be remembered that the Gram solution contains iodine as
the complex ion Is, whereas it eists in alcohol in molecular form.
1 A similar test with practically water-free ethyl alcohol showed a solubility
below 0.3 per cent. This explains why the Gram test cannot succesfully be
carried out under anhydrous conditions.
 THE GRAM STAIN AND DIFFERENTIAL STAINING 311

Moreover, the permeability of the cell in. water is probably differ-

ent than in alcohol.
If the above reasoning is valid, retention of the dye by positives
may be due not only to a low permeability to the dye, but to a low
permeability to iodine as well. Asan indication of the importance
of the latter factor may be cited our observation that the decolori-
zation of Gram-negatives is prevented by the addition of 0.2 to
0.3 per cent of iodine to the alcohol. Still more striking is the
fact that methyl alcohol, which decolorizes most Gram-positives
almost instantly, does not, when as little as 0.01 per cent iodine is
TABLE 1
PER CZNT IODINE
ORGANISM _
0.01 0.02 0.04 0.06 0.08
B. subtili .+ s + + + +
M. tetragenus .. + + + +

Sarcina aurantiaca .................. -} + - + + +

Staph. aureus .. +:} - + +

Strep. hemolyticus.ou _ _
-

- i
D. pneumoniae ..............j.+ +

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added, decolorize such positives as B. subtilis, M. tetragenus, etc.,
while only slightly larger additions of iodine are necessary to pre-
vent decolorization of other positives.
Table 1 shows the results of tests on six organisms obtained
from Bellevue Medical School.2 A set of slides was prepared,
on each of which were placed, side by side, the six organisms.
After staining with a 0.5 per cent solution of crystal violet in 2 per
cent sodium acetate, they were treated with Gram iodine solu-
tion, washed and dried, and then placed in small jars containing
various concentrations of iodine in methyl alcohol (C. P. Eimer
These organisms were supplied to us through the kindness of Dr. Wm. H.
2
Park to whom we owe thanks.
JOURNAL OF BACTERIOLOGY, VOL. XXV, NO. 3
3121M. L. KAPLAN AND LEAH KAPLAN
and Amend) to which 5 per cent of water was added. Small
quantities of freshly prepared and dried calcium carbonate were
placed in each jar to insure neutrality of the methanol. The
slides were kept in motion in the solutions for two minutes and
then promptly washed and counterstained with Safranin for
twenty to thirty seconds.
We see from the table that Gram-positive bacteria may be dif-
ferentiated according to their degree of positivity as measured by
iodine concentration in methyl alcohol necessary to prevent ex-
traction of iodine and dye from the cell.
Similar tests with other organisms gave approximately constant
values for each species, so long as the water content of the alcohol
remained constant. The more aqueous the methanol, the more
iodine was required to prevent extraction.
Organisms were frequently encountered which showed varying
degrees of positivity in different parts of the cell body. Figure 1
is the photograph of a twelve-hour culture of a soil spore-bearer
which had been Gram stained and then treated as above with
methyl alcohol containing 0.04 per cent iodine. A strain of B.
diphtheriae isolated from a clinical case and tested in the same way
gave positive terminal metachromatic granules at 0.01 to 0.02
per cent iodine, while the rest of the cell body required much
higher iodine concentrations. It is interesting to note that a
strain of B. Hoffmani isolated from a nasal discharge required 0.05

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to 0.06 per cent iodine to prevent dye-iodine escape.
Whether this difference is due to a difference in permeability
to dye or to iodine is hard to say off hand, since a decrease in either
would probably have the same effect. In order to investigate
this point, we exposed crystal violet stained slides, omitting
Gram iodine treatment, to ethyl alcohol solutions of iodine, since,
under these conditions, iodine and dye move in opposite directions
and cell permeabilities to them have opposite effects. For exam-
ple, table 1 shows M. tetragenus to be more highly positive than
Sarcina aurantiaca. If the greater positivity were ascribable to
lower permeability to iodine, one would expect M. tetragenus to
require the higher concentration of iodine to insure dye retention.
The test, however, gave the opposite result. On the other hand,
 w*~ ~ .~
THE GRAM STAIN AND DIFFERENTIAL STAINING

B. subtilis, which shows the highest degree of positivity, does not

retain the dye in all parts of the cell body even when 0.2 per cent
iodine has been added to the alcohol, an amount which is sufficient
to suppress the escape of dye even from B. coli. The arrangement
of stained and unstained spots obtained in this way from certain

;.....
..:.:.
..... ..

M
evt
....~~~~~~~~~~~;T
/..

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

FIG. 1. TWELVE-HOUR CULTURE OF SOIL SPORE BEARER, GRAM-TREATED AND

EXPOSED TO THE ACTION OF METHYL ALCOHOL CONTAINING 0.04 PER
CENT IODINE

members of the subtilis group is striking. Figures 2 and 3 are

photographs of slides of B. subtilis and a soil spore bearer, respec-
,U.
.. :.. ...
........
::s:

b
,;:.:::

.* ;S.
313
3

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tively. The slides had been stained with crystal violet and then
treated for two minutes with 0.08 per cent cent iodine in 85 per
cent alcohol. The unequal behavior of different parts of the cell
was also observed in other organisms. Thus, B. diphtheriae
shows retention of dye in the terminal bodies, while B. Hoffmani,
in the early transplants after isolation from the human body,
showed decolorization on one side and dye retention on the other
side of the cell.
 ..

4
,F..* Pa. v
W.
't
,I,
,,

iA

FIG. 2. B. SUBTILIS STAINED WITH CRYSTAL VIOLET AND TREATED WITH 0.08 PER
CENT IODINE IN 85 PER CENT ETHYL ALCOHOL
Note stained and unstained spots. Compare with figure 3.

N '.::

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a.
ill ¶
'a
(a
a
Mw
¾.
r1
IV16 'I.4
't

FIG. 3. SOIL SPORE BEARER STAINED WITH CRYSTAL VIOLET AND TREATED WITH
0.08 PER CENT IODINE IN 85 PER CENT ETHYL ALCOHOL
314
 THE GRAM STAIN AND DIFFERENTIAL STAINING 315
These facts point to different degrees of permeability to iodine
of the Gram-positives. The comparative resistance of M. tetra-
genus to decolorization by ethyl alcohol containing iodine indi-
cates that its resistance to dye escape is higher than that of
Sarcina without a corresponding increased resistance to iodine
passage. This shows that the resistance is due not only to the
size of the moving molecule but also to factors specific to the cell.
Instances of a certain degree of parallelism between the two re-
sistances can be cited, though limited to interior parts of the cell
and aqueous iodine solutions. Treatment of bacteria with dilute
alkali (0.1 normal NaOH) developed a difference in different
parts of the cell which becomes pronounced by either of the follow-
ing staining methods: (1) Staining with methylene blue and then
covering with Gram iodine for one minute; (2) staining with
crystal violet and counterstaining with safranin for two minutes.
Figures 4 and 5 illustrate the first method applied to B. subtilis
and a soil spore-bearer respectively; and figures 6 and 7 show the
same organisms treated by the second method. The similarities
of the two photographs of each organism are striking, since the
dark bodies in the photographs. obtained by the first method are
due to the failure of iodine to penetrate to those parts, while in
those obtained by the second method they are due to the failure
of crystal violet to escape from those parts. We see here ex-
amples of parallelism between the two resistances. Another ex-
ample is the strain of B. diphtheriae mentioned above which, when

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stained by these two methods without previous alkali treatment,
gave good pictures of the terminal metachromatic granules
(see figure 8, from a photograph after staining this organism with
crystal violet and counterstaining with safranin for two minutes).
These granules also appear when Gram stained slides are treated
with iodine in methyl alcohol.
Such parallelisms, however, do not exclude the possibility of
divergence. The behavior of Gram-negatives may be regarded as
an extreme case. When suspended in water they often exhibit
greater resistance to dye escape than many positives. B. coli
stained with crystal violet and counterstained with safranin
(dye displacement method called method 2 above) lost its crystal
316 M. L. KAPLAN AND LEAH KAPLAN

...t ...

$s ' U .

S -F

* #,

j#

FIG. 4. B. SUBTILIS TREATED WITH 0.1 N NaOH, STAINED WITH METHYLENE BLUE,
AND DECOLORIZED WITH GRAM IODINE SOLUTION

t.twu
ir
Ep

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#d

.4*.U 6s
B.
- * ffi.
r
o 44
8*v,

FIG. 5. SOIL ORGANISM TREATED WITH 0.1 N NaOH, STAINED WITH METHYLENE
BLUE, AND DECOLORIZED WITH GRAM IODINE SOLUTION
 t
i
~-.
f$

*
. s' S E >
THE GRAM STAIN AND DIFFERENTIAL STAINING

F-

FIG. 6. B. SUBTILIS TREATED WITH 0.1 N NaOH, STAINED WITH CRYSTAL VIOLET
AND COUNTERSTAINED WITH SAFRANIN FOR Two MINUTES

4L

it
:

iiiUt
<
NsM

...'4':~~~~~~~~~~~~~~~~~~~~.....
.:~~~~A T

q ::' .'R.,' . b. .',.....M.

A~~~~~~J

@
'..' :'

FIG. 7. SOIL SPORE BEARER TREATED WITH 0.1 N NaOH, STAINED WITH CRYSTAL
VIOLET, AND COUNTERSTAINED WITH SAFRANIN FOR Two MINUTES
.RZ
I

i.:k.
z," I

I
317

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318 M. L. KAPLAN AND LEAH KAPLAN

violet less readily than Sarcina and many soil spore bearers.
Since the quantity of the counterstain is great compared to the
originally absorbed dye, preferential affinities are not likely to
play any role. We can conclude, therefore, that B. coli offers
considerable resistance to dye passage. Since B. coli and other
Gram-negatives stained with crystal violet and treated with iodine
according to the Gram method show remarkable acid-fastness, the

FI D C Ve

4t Of ~ : 1

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WITH SAFRANIN FOR Two MINUTES

possibility of a failure of iodine to enter, as suggested by Brudny,

is excluded. From this we conclude that Gram negativity is due
to low resistance to iodine escape (that is, high permeability to
iodine). Attempts to measure this resistance by iodine back-
pressure, so effective in the case of the Gram-positives, did not
give clearly marked results. B. coli and others tested with low
iodine concentrations in ethyl alcohol showed retention of the dye
only in a thin line in the interior of the cell. As the iodine concen-
 THE GRAM STAIN AND DIFFERENTIAL STAINING 319
tration is increased, the line grows in thickness until it reaches the
outer visible border of the cell at about 0.2 per cent iodine. Many
Gram-positives when subjected to the action of methyl alcohol
containing iodine in insufficient amount to prevent the escape of
the dye completely, show, in contrast to the Gram-negatives, some
decolored cells and some cells in which the dye is retained.
The lack of a sharp transition in the case of the negatives, and the
contrasting behavior of the positives, gives the impression that the
latter have a limiting membrane of higher resistance or, to use
Churchman's phraseology (1929), a cortex, which the Gram-nega-
tives lack.
Another indication of a structural difference between Gram-
positives and negatives is the ease with which the latter are dis-
solved by alkali in contrast to the resistance offered by the former
(Smith, 1922). We have observed that even 0.1 N sodium hy-
droxide converts emulsions of Gram-negatives into slimy masses.
Some members of the group, such as B. typhosus, Proteus and
others have formed slime when treated with alkali as dilute as
0.025 N. We have encountered an exception to this rule in the
case of an unidentified borderline organism which formed slime
with 0.2 N alkali and yet, in view of its behavior when Gram-
treated and immersed in ethyl alcohol containing low concentra-
tions of iodine, should be classed with the positives. Despite this
exception, the only one encountered, we consider this slime forma-
tion to be a fair test for Gram negativity.

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The test is performed by emulsifying scrapings from an agar
culture on a slide in a drop of water and mixing a small loopful
of the emulsion with an equal quantity of 0.2 N NaOH on another
slide. B. proteus, B. typhosus, and para A and B are converted
almost instantly into a heavy, sticky mass; while B. coli some-
times requires a few seconds for complete conversion. The alkali
treatment usually does not interfere with ordinary staining, and
brings about the differentiation of the interior parts which be-
comes visible as beading when treated by methods 1 or 2 de-
scribed above. In the cases of B. coli treated with quantities of
alkali small in proportion to the number of organisms present,
320 M. L. KAPLAN AND LEAH KAPLAN

methylene blue and erythrosin are effective in bringing out the

beading, the body appearing faintly pink while the beads take on a
deep blue.
In general, the combination of a basic dye such as methylene
blue and an acid dye such as eosin or erythrosin has been found to
possess excellent differentiation possibilities, though dividing
bacteria along entirely new lines. Thus, B. subtilis and other
spore bearers lose the initial blue basic dye and become pink, with
occasional round blue bodies in the interior of the cells. Gram-
negatives, on the other hand, retain the original blue dye, although
B. proteus and B. typhosus give a mixture of blue and pink cells.
All the streptococci we could obtain stained pink; while the staphy-
lococci varied, some staining entirely blue while others showed
mixtures of pink and blue cells. Previous treatment of smears
with sufficient quantities of alkali resulted in Gram-negatives
staining pink exclusively, while blue-staining Gram-positives
gave mixtures of blue and pink cells. Acid treatment prior to
staining also affects different organisms differently. In short,
there seem to be considerable possibilities of differentiating bac-
teria by means of dye pairs.
It should be noted that the capacity of cells to retain methylene
blue after treatment with erythrosin is completely lost if smears
are treated with methyl alcohol, which fact points to the removal
of some important bacterial constituent from the cell, and elimi-

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nates the possibility of fixing with alcohol.
Since most Gram-negative bacteria retain the original blue
dye, the method described above should be advantageous in
searching for these organisms in smears containing pus. We
tried it on a pus-containing nasal discharge of a child sick with the
grippe and were able to see numerous small blue, stained bacilli,
probably of the influenza type, against a pink background of
mucus and pus cells.
Alluring as the prospects of research in this direction appear,
the lack of a sufficient number of labelled bacteria and a proper
collection of dyes has prevented our continuing along these lines.
We are at present investigating the effect of solutions of iodine in
other solvents.
 THE GRAM STAIN AND DIFFERENTIAL STAINING 321
SUMMARY
Gram-positives act in a manner indicating a low permeability
to iodine in alcoholic solution. This, combined with the low
alcohol solubility of the dye-iodine compound, is probably the
cause of dye retention by positives in the Gram test. A method
is described for measuring the degree of permeability to alcoholic
iodine. This makes possible the subdivision of Gram-positives
into groups according to their degree of permeability. In con-
trat to the Gram-negatives, positives are shown to have proper-
ties indicating a high resistance to iodine passage along the outer
limits of the cell, in harmony with the view of Benians (1919-20).
This assumption is further supported by differences in behavior
on alkali treatment, which readily converts negatives into slime.
Although positives do not form slime under these conditions,
both types give rise to beading when treated with alkali. Meth-
ods are described for bringing out this beading. Alkali treatment
resulting in slime formation does not interfere with ordinary
staining reactions. The method described, of studying resistance
to dye movement by displacing one basic dye with another, while
often differentiating bacteria of the same group, does not show a
parallelism between resistance to dye movement and Gram posi-
tivity. Remarkable results in differentiation of bacteria along
new lines were obtained by the dye replacement method when an
acid dye was used to replace a basic dye. These results with dye
pairs indicate further possibilities.

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REFERENCES
BENIANS 1912 Jour. Path. and Bacteriol. 17, 199.
BENIANS 1919-20 Jour. Path. and Bacteriol., 23, 411.
BRUDNY 1908 Central. f. Bakteriol., 21, 62.
BURKE AND BARNES 1929 Jour. Bacteriol., 18, 69.
CHURCHMAN 1929 Jour. Bacteriol., 18, 413.
SMITH, H. 1922 Amer. Jour. Hyg., 2, 607.
STEARN AND STEARN 1930 Jour. Bacteriol., 20, 287.