Gram stain or Gram staining, also called Gram's method, is a method of
staining used to distinguish and classify bacterial species into two
large groups (gram-positive and gram-negative). The name comes from
the Danish bacteriologist Hans Christian Gram, who developed the
Gram staining differentiates bacteria by the chemical and physical
properties of their cell walls by detecting peptidoglycan, which is
present in the cell wall of
Gram-positive bacteria. Gram-negative
cells also contain peptidoglycan, but a very small layer of it that is
dissolved when the alcohol is added. This is why the cell loses its
initial color from the primary stain.
Gram-positive bacteria retain
the crystal violet dye, and thus are stained violet, while the
Gram-negative bacteria do not; after washing, a counterstain is added
(commonly safranin or fuchsine) that will stain these Gram-negative
bacteria a pink color. Both
Gram-positive bacteria and Gram-negative
bacteria pick up the counterstain. The counterstain, however, is
Gram-positive bacteria because of the darker crystal violet
Gram stain is almost always the first step in the preliminary
identification of a bacterial organism. While Gram staining is a
valuable diagnostic tool in both clinical and research settings, not
all bacteria can be definitively classified by this technique. This
gives rise to gram-variable and gram-indeterminate groups.
3.3 Gram variable and gram-indeterminate bacteria
4 Orthographic note
5 See also
7 External links
The method is named after its inventor, the Danish scientist Hans
Christian Gram (1853–1938), who developed the technique while
Carl Friedländer in the morgue of the city hospital in
Berlin in 1884. Gram devised his technique not for the purpose of
distinguishing one type of bacterium from another but to make bacteria
more visible in stained sections of lung tissue. He published his
method in 1884, and included in his short report the observation that
the typhus bacillus did not retain the stain.
Gram staining is a bacteriological laboratory technique used to
differentiate bacterial species into two large groups (gram-positive
and gram-negative) based on the physical properties of their cell
walls.[page needed] Gram staining is not used to classify
archaea, formerly archaeabacteria, since these microorganisms yield
widely varying responses that do not follow their phylogenetic
Gram stain is not an infallible tool for diagnosis,
identification, or phylogeny, and it is of extremely limited use in
environmental microbiology. It is used mainly to make a preliminary
morphologic identification or to establish that there are significant
numbers of bacteria in a clinical specimen. It cannot identify
bacteria to the species level, and for most medical conditions, it
should not be used as the sole method of bacterial identification. In
clinical microbiology laboratories, it is used in combination with
other traditional and molecular techniques to identify bacteria. Some
organisms are gram-variable (meaning they may stain either negative or
positive); some are not stained with either dye used in the Gram
technique and are not seen. In a modern environmental or molecular
microbiology lab, most identification is done using genetic sequences
and other molecular techniques, which are far more specific and
informative than differential staining.
Gram staining has been suggested to be as effective a diagnostic tool
as PCR in one primary research report regarding gonococcal
urethritis.[non-primary source needed]
Gram-negative bacterial infection and Gram-positive
Gram stains are performed on body fluid or biopsy when infection is
suspected. Gram stains yield results much more quickly than culturing,
and is especially important when infection would make an important
difference in the patient's treatment and prognosis; examples are
cerebrospinal fluid for meningitis and synovial fluid for septic
Gram-positive bacteria have a thick mesh-like cell wall made of
peptidoglycan (50–90% of cell envelope), and as a result are stained
purple by crystal violet, whereas gram-negative bacteria have a
thinner layer (10% of cell envelope), so do not retain the purple
stain and are counter-stained pink by safranin. There are four basic
steps of the Gram stain:
Applying a primary stain (crystal violet) to a heat-fixed smear of a
Heat fixation kills some bacteria but is mostly
used to affix the bacteria to the slide so that they don't rinse out
during the staining procedure
The addition of iodide, which binds to crystal violet and traps it in
Rapid decolorization with ethanol or acetone
Counterstaining with safranin.
Carbol fuchsin is sometimes
substituted for safranin since it more intensely stains anaerobic
bacteria, but it is less commonly used as a counterstain.
Gram stain procedure and effect on
Gram-positive cell walls.
Crystal violet (CV) dissociates in aqueous solutions into CV+ and
chloride (Cl−) ions. These ions penetrate through the cell wall and
cell membrane of both gram-positive and gram-negative cells. The CV+
ion interacts with negatively charged components of bacterial cells
and stains the cells purple.
Iodide (I− or I−
3) interacts with CV+ and forms large complexes of crystal violet and
iodine (CV–I) within the inner and outer layers of the cell. Iodine
is often referred to as a mordant, but is a trapping agent that
prevents the removal of the CV–I complex and, therefore, color the
When a decolorizer such as alcohol or acetone is added, it interacts
with the lipids of the cell membrane. A gram-negative cell loses
its outer lipopolysaccharide membrane, and the inner peptidoglycan
layer is left exposed. The CV–I complexes are washed from the
gram-negative cell along with the outer membrane.
In contrast, a gram-positive cell becomes dehydrated from an ethanol
treatment. The large CV–I complexes become trapped within the
gram-positive cell due to the multilayered nature of its
peptidoglycan. The decolorization step is
critical and must be timed correctly; the crystal violet stain is
removed from both gram-positive and negative cells if the decolorizing
agent is left on too long (a matter of seconds).
After decolorization, the gram-positive cell remains purple and the
gram-negative cell loses its purple color.
Counterstain, which is usually positively charged safranin or basic
fuchsine, is applied last to give decolorized gram-negative bacteria a
pink or red color.
Gram-positive bacteria generally have a single membrane (monoderm)
surrounded by a thick peptidoglycan. This rule is followed by two
Firmicutes (except for the classes
Negativicutes) and the Actinobacteria. In contrast, members of
the Chloroflexi (green non-sulfur bacteria) are monoderms but possess
a thin or absent (class Dehalococcoidetes) peptidoglycan and can stain
negative, positive or indeterminate; members of the
Deinococcus-Thermus group, stain positive but are diderms with a thick
peptidoglycan.[page needed] 
Historically, the gram-positive forms made up the phylum Firmicutes, a
name now used for the largest group. It includes many well-known
genera such as Bacillus, Listeria, Staphylococcus, Streptococcus,
Enterococcus, and Clostridium. It has also been
expanded to include the Mollicutes, bacteria like
Mycoplasma that lack
cell walls and so cannot be stained by Gram, but are derived from such
Some bacteria have cell walls which are particularly adept at
retaining stains. These will appear positive by
Gram stain even though
they are not closely related to other gram-positive bacteria. These
are called acid fast bacteria, and can only be differentiated from
other gram-positive bacteria by special staining
Gram-negative bacteria generally possess a thin layer of peptidoglycan
between two membranes (diderms). Most bacterial phyla are
gram-negative, including the cyanobacteria, spirochaetes, and green
sulfur bacteria, and most
Proteobacteria (exceptions being some
members of the
Rickettsiales and the insect-endosymbionts of the
Gram variable and gram-indeterminate bacteria
Some bacteria, after staining with the Gram stain, yield a
gram-variable pattern: a mix of pink and purple cells are
seen. In cultures of Bacillus, Butyrivibrio, and
Clostridium, a decrease in peptidoglycan thickness during growth
coincides with an increase in the number of cells that stain
gram-negative. In addition, in all bacteria stained using the Gram
stain, the age of the culture may influence the results of the
Gram-indeterminate bacteria do not respond predictably to Gram
staining and, therefore, cannot be determined as either gram-positive
or gram-negative. Examples include many species of Mycobacterium,
including M. tuberculosis and M. leprae.
The term Gram staining is derived from the surname of Hans Christian
Gram, the eponym (Gram) is therefore capitalized but not the common
noun (stain) as is usual for scientific terms. The adjectives
'gram-positive' and 'gram-negative'; as eponymous adjectives, their
initial letter can be either lowercase 'g' or capital 'G', depending
on whose style guide (if any) governs the document being written.
Lowercase style is used by the US Centers for Disease Control and
Prevention and other style regimens such as the AMA style.
Dictionaries may use lowercase, uppercase, or
both. Uppercase 'Gram-positive' or 'Gram-negative' usage is
also common in many scientific journal articles and
publications. When articles are submitted to journals,
each journal may or may not apply house style to the postprint
Preprint versions contain whichever style the author happened
to use. Even style regimens that use lowercase for the adjectives
'gram-positive' and 'gram-negative' still use capital for 'Gram
Bacterial cell structure
^ John G. Holt; Noel R. Krieg; Peter H.A. Sneath; James T. Staley;
Stanley T. Williams (1994). Bergey's Manual of Determinative
Bacteriology (9th ed.). Lippincott Williams & Wilkins. p. 11.
^ Austrian, R. (1960). "The
Gram stain and the etiology of lobar
pneumonia, an historical note". Bacteriological Reviews. 24 (3):
261–265. PMC 441053 . PMID 13685217.
^ Gram, H.C. (1884). "Über die isolierte Färbung der Schizomyceten
in Schnitt- und Trockenpräparaten". Fortschritte der Medizin (in
German). 2: 185–189. .
English translation in: Brock, T.D. (1999). Milestones in Microbiology
1546–1940 (2 ed.). ASM Press. pp. 215–218.
Translation is also at: Brock, T.D. "Pioneers in Medical Laboratory
Science: Christian Gram 1884". Hoslink. Retrieved 2010-07-27.
^ a b Ryan K.J., Ray C.G. (editors) (2004). Sherris Medical
Microbiology (4th ed.). McGraw Hill. pp. 232f.
ISBN 0838585299. CS1 maint: Extra text: authors list (link)
[full citation needed]
^ a b c d Madigan, M.T.; Martinko J.; Parker J. (2004). Brock Biology
of Microorganisms (10th ed.). Lippincott Williams & Wilkins.
ISBN 0-13-066271-2. [full citation needed]
^ Beveridge T.J. (2001). "Use of the
Gram stain in microbiology".
Biotechnic & Histochemistry. 76 (3): 111–118.
doi:10.1080/714028139. PMID 11475313.
^ El-Garnal, A.H., Al-Otaibi, S.R., Alshamali, A., Abdulrazzaq, A.,
Najem, N., and Fouzan, A.A.
Polymerase chain reaction
Polymerase chain reaction is no better
Gram stain for diagnosis of gonococcal urethritis. Indian Journal
of Dermatology, Venereology, and Leprology, (2009); 75,
101.[non-primary source needed]
^ Søgaard M.; Nørgaard M.; Schønheyder H. (2007). "First
notification of positive blood cultures: high accuracy of the Gram
stain report". Journal of Clinical Microbiology. 45 (4): 1113–1117.
doi:10.1128/JCM.02523-06. PMC 1865800 .
^ Microbiology: Principles and Explorations, p 65; Jacquelyn.G. Black
Prentice Hall, 1993.
^ "Medical Chemical Corporation". med-chem.com. Retrieved 9 March
^ Leboffe, Michael (2014). Microbiology
Laboratory Theory and
Application (3rd ed.). Englewood, CO: Morton Publishing Company.
p. 105. ISBN 1617312800.
File - Stain theory - What a mordant is not".
stainsfile.info. Retrieved 9 March 2016.
^ "Welcome to Microbugz - Gram Stain". www.austincc.edu. Retrieved
^ a b c Tim,, Sandle,. Pharmaceutical microbiology : essentials
for quality assurance and quality control. ISBN 9780081000229.
^ a b Gram's Serendipitous Stain by Hardy's Diagnostics at
^ Beveridge T.J.; Davies J.A. (November 1983). "Cellular responses of
Bacillus subtilis and
Escherichia coli to the Gram stain". Journal of
Bacteriology. 156 (2): 846–58. PMC 217903 .
^ Davies J.A.; Anderson G.K.; Beveridge T.J.; Clark H.C. (November
1983). "Chemical mechanism of the
Gram stain and synthesis of a new
electron-opaque marker for electron microscopy, which replaces the
iodine mordant of the stain". Journal of Bacteriology. 156 (2):
837–845. PMC 217902 . PMID 6195147.
^ a b c Don J. Brenner, Noel R. Krieg, James T. Staley (July 26, 2005)
. George M. Garrity, ed. Introductory Essays. Bergey's Manual of
Systematic Bacteriology. 2A (2nd ed.). New York: Springer.
p. 304. ISBN 978-0-387-24143-2. British Library no.
GBA561951. CS1 maint: Uses authors parameter (link)
^ Galperin, Michael Y. (2013-12-27). "Genome Diversity of
Spore-Forming Firmicutes". Microbiology Spectrum. 1 (2).
doi:10.1128/microbiolspectrum.tbs-0015-2012. ISSN 2165-0497.
PMC 4306282 . PMID 26184964.
^ Practical Medical Microbiology by Dr. Hams H. Hashem, from
^ "THE ACID FAST STAIN". www2.highlands.edu. Retrieved
^ Beveridge, Terry J. (March 1990). "Mechanism of gram variability in
select bacteria". Journal of Bacteriology. 172 (3): 1609–20.
doi:10.1128/jb.172.3.1609-1620.1990. PMC 208639 .
^ Beveridge, Terry J. (March 1990). "Mechanism of Gram Variability in
Select Bacteria" (PDF). Journal of Bacteriology. 172 (3): 1609–20.
doi:10.1128/jb.172.3.1609-1620.1990. PMC 208639 .
PMID 1689718. Retrieved 17 September 2016.
^ Black, Jacquelyn (2012). Microbiology: Principles and Exploration
(8th ed.). John Wiley & Sons. p. 68.
^ Reynolds J.; Moyes R.B.; Breakwell D.P. (2009). "Differential
staining of bacteria: acid fast stain". Current Protocols in
Microbiology. Appendix 3: Appendix 3H.
doi:10.1002/9780471729259.mca03hs15. PMID 19885935.
^ Waddingham, Anne (28 August 2014). New Hart's Rules: The Oxford
Style Guide. OUP Oxford. p. 105. ISBN 978-0199570027.
^ Centers for Disease Control and Prevention. Emerging Infectious
Diseases Journal Style Guide. Preferred Usage
^ Elsevier, Dorland's Illustrated Medical Dictionary, Elsevier.
^ Merriam-Webster, gram–positive, Merriam-Webster.
^ "Definition of Gram-positive". Collins.
^ "Gram stain". Oxford Dictionary.
^ "Definition of Gram-positive". Medicinenet.
^ "Gram negative/positive". Business dictionary.
^ "gram-pos·i·tive or Gram-pos·i·tive". The American Heritage
^ a b "Gram-positive". Dictionary.com.
^ Lisa Brown, Julie M. Wolf, Rafael Prados-Rosales & Arturo
Casadevall (2015). "Through the wall: extracellular vesicles in
Gram-positive bacteria, mycobacteria and fungi". Nature Reviews
Microbiology. 13: 620–630. doi:10.1038/nrmicro3480.
PMC 4860279 . PMID 26324094. CS1 maint: Uses authors
^ Kristen L. Mueller (12 June 2015). "Detecting Gram-negative
bacteria". Science. 348 (6240): 1218.
Wikimedia Commons has media related to Gram stains.
The Wikibook School Science has a page on the topic of: Gram staining
Gram staining technique video
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