Technical Information Sheet No.9
A STANDARDIZED GRAM STAINING PROCEDURE
Dr. Dieter Claus
Information Centre for European Culture Collections,
Mascheroder Weg 1b, D-3300 Braunschweig, Germany
Introduction
When bacteria are stained with certain basic dyes and treated with iodine some
species can be easily decolourized with organic solvents, such as ethanol or
acetone, whereas others resist decolourization. These characteristics were first
observed by H.C.J. Gram, a Danish physician, about a century ago. From that
time, bacteria that retain the stain have been said to be Gram-positive, those
that are decolourized are called Gram-negative. Although Gram failed to
recognize the taxonomic value of his staining procedure, by the end of the
nineteenth century it was generally realized that these staining characteristics
correlated with important physiological and chemical characteristics of the cell.
Today, the Gram reaction is still a character of fundamental importance in
bacterial classification and identification (Barthomolew & Mittwer 1952). It is
one of the most essential of the genus criteria. It is said that the most frequent
causes of incorrect identification of bacteria are errors in the determination of
shape, motility and Gram reaction. The Gram staining reaction observed with a
bacterial strain does not necessarily correspond to its 'Gram type', a term
proposed by Wiegel (1981) to indicate the classification of bacteria into
taxonomically relevant groups. Thus, certain Gram-positive species or genera
include strains described as Gram-negative, and some bacteria, placed in a
Gram-negative taxon, show a tendency to resist decolourization and have a
more or less Gram-positive appearance.
The Gram staining reaction, therefore, may be misleading, both for
classification and for proper identification. A 'false negative' or a 'false positive'
staining reaction may be due to (1) the properties of the organism itself, (2) the
age of the culture or (3) the method applied.
1. It is not yet fully understood why some organisms give a Gram reaction
which does not correspond to their Gram type, based on other
biochemical or structural properties.
2. Some Gram-positive bacteria appear Gram-negative when they have
reached a certain age, varying from a few hours to a few days. On the
other hand, some Gram-negative bacteria may become Gram-positive in
older cultures. For this reason it is strongly recommended to use very
young cultures for the staining procedure, after growth has become just
visible.
3. Since the original procedure of Gram, many variations of the Gram
staining technique have been published. Some of them have improved
the method, others include some minor technical variants of no value.
Bartholomew (1962) has pointed out that each variation in the Gram
staining procedure has a definite limit to its acceptability. Any final result
is the outcome of the interaction of all of the possible variables.
Gram Staining Reagents
Hucker's crystal violet reagent
Solution A
Solution B
Crystal violet
2.0 g
Ammoniumoxalate
0.8 g
Ethanol (95%)
20.0 ml
Distilled water
80.0 ml
Mix solution A and B. This mixture is stable and can be kept at room
temperature for months.
Stabilized Lugol- PVP complex
Iodine
KI
Polyvinylpyrrolidone
Add distilled water to 100ml
Procedure
Counterstain
1.3
Safranin 0
g
2.0 Ethanol
g (95%)
1.0 Distilled
g water
0.25
g
10.0
ml
100.0
ml
1. Prepare a light suspension of cells from very young cultures grown on
appropriate agar medium. If the suspension prepared is too turbid, dilute with
distilled water.
2. Add one drop to a clean glass slide and spread the drop with a loop over the
surface of the slide. Allow to air-dry.
3. Cover the slide with methanol and allow to evaporate at room temperature.
4. Flood the slide for 1 min with Hucker's reagent.
5. Wash for 5 s by dipping the slide into tap water in a 250-ml beaker. The tap
water in this beaker should be constantly replaced by a stream of water
running into the beaker at a rate of about 30ml per sec.
6. Rinse off the excess water with stabilized PVP-iodine-KI solution, then flood
the slide with fresh iodine solution for 1 min.
7. Wash the slide for 5 s in water, as described under (5).
8. Decolourize the wet slide by immersing it for 1min in each of three Coplin
jars containing n-propanol. Agitate the slide while it is in the decolourizer.
Replace the propanol in the first jar after every 10 slides. Move the remaining
two jars up in sequence. Refill the empty jar and place it last.
9. Wash the slide for 5 s in water, as described under (5).
10. Rinse off the excess water with the counterstain, then flood with fresh
counterstain for 1min. The counterstain may be omitted.
11. Wash the slide for 5 s in water, as described under (5).
12. Allow the slide to air-dry.
13. Repeat the procedure with control organisms (Gram-negative: Escherichia
coli, Gram-positive: Micrococcus luteus) which also can be used as a mixed cell
suspension.
14. Examine the preparations with the oil immersion objective of the bright
field microscope (do not use the phase-contrast objective!)
15. Gram-positive cells appear purple and Gram-negative cells pink. (The iris of
the microscope condenser should be opened as wide as possible. With a closed
condenser colours can hardly be discriminated.)
Precautions
Some factors which are important when determining the Gram reaction of an
organism include the following:
1. Smears must be prepared in such a way that cells lie separately (about 100
cells per microscopic field as overcrowding prevents proper decolourization) .
2. The degree of Gram-positivity of cells can be influenced considerably by the
method of fixation. The common practice of heat fixation of smears before
Gram staining may cause Gram-positive cells to stain Gram-negatively.
Methanol fixation has proved to be the most reliable technique. Gram-positive
bacteria fixed by methanol are more resistant to decolourization than fixed by
any other technique (Magee et al. 1975).
3. Stock solutions of I2-KI in water are unstable. The degree to which iodine is
lost is influenced by temperature and other factors. As the concentration of
iodine in the mordant solution decreases, bacterial smears become more
susceptible to decolourization. Therefore, I2 -KI solutions should be stored
below 25C in a closed bottle for not longer than 3 weeks.
The problem of loss of iodine can be overcome by employing
polyvinylpyrrolidine (PVP) in the I2 -KI solution. PVP complexes with iodine. The
complex is stable and has a long shelf life. Cells treated with the stabilized
Lugol-PVP complex exhibit the same degree of Gram-positivity as those treated
with a freshly prepared solution of I2 -KI.
4. Gram stained preparations have to be observed with bright-field optics.
Phase-contrast microscopy does not allow the recognition of true colours.
Gram-positive bacteria may be seen under phase-contrast as red cells. Using
bright-field optics, Gram-positive cells are purple or blue and Gram-negative
pink due to counterstain with safranin. With bright-field optics colours can be
discriminated best if the condenser iris is opened as far as possible without
discomfort to the eyes.
5. Bacteria which stain only weakly Gram-positive are best detected if the
preparation is not counterstained with safranin. In this case, the cells of the
preparation can be easily detected using phase-contrast microscopy.
Thereafter, bright-field illumination is applied to decide on the colour of the
cells.
6. The Gram staining procedure does not always give clear-cut results. Some
organisms are Gram-variable and may appear either Gram-negative or Grampositive according to the conditions. With these types of organisms, Gram-
positive and Gram-negative cells may be present within the same preparation.
To determine if an organism belongs to this variable group, it is necessary that
it is stained at two or three different ages (very young cultures should be used).
If an organism changes from positive to negative or vice versa during its
growth cycle, this change should be recorded with a statement as to the age of
the culture when the change was first observed. In case a Gram-variable
reaction is observed it is also good to check the purity of the culture.
7. For all staining procedures, grease-free slides should be used. On a clean
slide a drop of water will spread out in a thin, uniform film. A greasy slide will
cause water to run in droplets, leading to badly stained preparations. New
slides generally are not clean enough for staining. For cleaning the slides
should be kept in alkaline KMnO4 solution and washed with distilled water.
Protect your fingers by using forceps while handling the slides. Also protect
your bench by using a slide-supporting sink rack.
REFERENCES
Barthomolew, J.W. & Mittwer, T. The Gram stain. Bacteriological Review 16, 1-29
(1952)
Barthomolew, J.W. Variables influencing results, and the precise definition of
steps in Gram staining as a means of standardizing the results obtained. Stain
Technology 37, 139-155. (1962)
Magee, Ch. M., Rodeheaver, G., Edgerton, M.T. & Edlich, R.F. A more reliable
Gram stainng technique diagnosis of surgical infections. American Journal of
Surgery 130, 341-346. (1975)
Wiegel, J. Distinction between the Gram reaction and the gram type of bacteria.
International Journal of Systematic Bacteriology 31, 88. (1981)
Published by : UNESCO / WFCC-Education Committee 1991
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