23-Oct-10 Gram staining - Wikipedia, the free e…

Gram staining
From Wikipedia, the free encyclopedia

Gram staining (or Gram's method) is an empirical method of

differentiating bacterial species into two large groups (Gram-positive and
Gram-negative) based on the chemical and physical properties of their
cell walls.[1] The Gram stain is almost always the first step in the
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, thus forming Gram variable
and Gram indeterminate groups as well.
Gram-positive anthrax bacteria
The word Gram is always spelled with a capital, referring to Hans (purple rods) in cerebrospinal fluid
Christian Gram, the inventor of Gram staining. sample. If present, a Gram-negative
bacterial species would appear pink.
(The other cells are white blood
cells).
Contents
1 History
2 Uses
2.1 Medical
2.2 Staining mechanism
3 Examples
3.1 Gram-negative bacteria
3.2 Gram-positive bacteria
4 See also A Gram stain of mixed
5 References Staphylococcus aureus (Gram
6 External links positive cocci) and Escherichia coli
(Gram negative bacilli)

History
The method is named after its inventor, the Danish scientist Hans Christian Gram (1853–1938), who developed the
technique while working with Carl Friedländer in the morgue of the city hospital in Berlin. Gram devised his
technique not for the purpose of distinguishing one group of bacteria from another but to enable bacteria to be seen
more readily in stained sections of lung tissue.[2] He published his method in 1884, and included in his short report
the observation that the Typhus bacillus did not retain the stain.[3]

Uses
Gram staining is used to differentiate bacterial species into two large groups (Gram-positive and Gram-negative)
based on the physical properties of their cell walls. Gram staining is not used to classify archaea, since these
en.wikipedia.org/wiki/Gram_staining 1/5
based on the
23-Oct-10 physical properties of theirGram
cell walls.
stainingGram stainingthe
- Wikipedia, is not
freeused
e… to classify archaea, since these

microorganisms yield widely varying responses that do not follow their phylogenetic groups.[4]

Gram staining is a bacteriological laboratory technique.[5] The technique is used as a tool for the differentiation of
Gram-positive and Gram-negative bacteria, as a first step to determine the identity of a particular bacterial
sample.[6]

The Gram stain is not an infallible tool for diagnosis, identification, or phylogeny, however. It is of extremely limited
use in environmental microbiology, and has been largely superseded by molecular techniques even in the medical
microbiology lab. Some organisms are Gram-variable (that means, they may stain either negative or positive); some
organisms are not susceptible to either stain used by the Gram technique. 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 information-rich than differential staining.

Medical

Gram stains are performed on body fluid or biopsy when infection is suspected. It yields results much more quickly
than culture, 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 arthritis.[5][7]

Being able to identify bacteria by stain result/ photo, or negative stain and history is a typical Medical Boards
question. Special Stains and Growth Media, such as India Ink for Cryptococcus and actual appearance of
hemolytic streps on growth plate are seen on test questions.

Staining mechanism

Gram-positive bacteria have a thick mesh-like cell wall made of peptidoglycan (50-90% of cell wall), which stains
purple while Gram-negative bacteria have a thinner layer (10% of cell wall), which stains pink. Gram-negative
bacteria also have an additional outer membrane which contains lipids, and is separated from the cell wall by the
periplasmic space. There are four basic steps of the Gram stain, which include applying a primary stain (crystal
violet) to a heat-fixed (death by heat) smear of a bacterial culture, followed by the addition of a trapping agent
(Gram's iodine), rapid decolorization with alcohol or acetone, and counterstaining with safranin.[8] Basic fuchsin is
sometimes substituted for safranin since it will more intensely stain anaerobic bacteria but it is much less commonly
employed as a counterstain.[9]

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.

Iodine (I – or I3 – ) 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 cell.[10]

When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane. A gram-
negative cell will lose 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

en.wikipedia.org/wiki/Gram_staining 2/5
23-Oct-10 Gram staining - Wikipedia, the free e…
timed correctly; the crystal violet stain will be 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 fuchsin, is applied last to give decolorized gram-
negative bacteria a pink or red color.[11][12]

Some bacteria, after staining with the Gram stain, yield a Gram-variable pattern: a mix of pink and purple cells are
seen. The genera Actinomyces, Arthobacter, Corynebacterium, Mycobacterium, and Propionibacterium have
cell walls particularly sensitive to breakage during cell division, resulting in Gram-negative staining of these Gram-
positive cells. 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.[13] In addition, in all bacteria
stained using the Gram stain, the age of the culture may influence the results of the stain.

Examples
Gram-negative bacteria

Main article: Gram-negative bacteria

The proteobacteria are a major group of Gram-negative bacteria. Other notable groups of Gram-negative bacteria
include the cyanobacteria, spirochaetes, green sulfur and green non-sulfur bacteria.

These also include many medically relevant Gram-negative cocci, bacilli and many bacteria associated with
nosocomial infections.

Gram-positive bacteria

Main article: Gram-positive bacteria

In the original bacterial phyla, 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 forms.

See also
Bacterial cell structure
Staining (biology)
Trichrome stain

References
1. ^ Bergey, David H.; John G. Holt; Noel R. Krieg; Peter H.A. Sneath (1994). Bergey's Manual of Determinative
Bacteriology (9th ed.). Lippincott Williams & Wilkins. ISBN 0-683-00603-7.
2. ^ Austrian, R. (1960). "The Gram stain and the etiology of lobar pneumonia, an historical note"
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC441053/pdf/bactrev00126-0009.pdf) . Bacteriol. Rev. 24 (3):
en.wikipedia.org/wiki/Gram_staining 3/5
23-Oct-10 Gram staining - Wikipedia, the free e…
261–265. PMID 13685217 (http://www.ncbi.nlm.nih.gov/pubmed/13685217) . PMC 441053
(http://www.pubmedcentral.gov/articlerender.fcgi?tool=pmcentrez&artid=441053) .
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC441053/pdf/bactrev00126-0009.pdf.
3. ^ Gram, HC (1884). "Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten" (in
German). Fortschritte der Medizin 2: 185–189.
English translation in: Brock, T.D. (1999). Milestones in Microbiology 1546-1940 (http://books.google.com/?
id=q5JHcs8w21gC&lpg=PP1&dq=Milestones%20in%20Microbiology&pg=PA215#v=onepage&q) (2 ed.). ASM
Press. pp. 215–218. ISBN 1555811426. http://books.google.com/?
id=q5JHcs8w21gC&lpg=PP1&dq=Milestones%20in%20Microbiology&pg=PA215#v=onepage&q.
Translation is also at: Brock, T.D.. Pioneers in Medical Laboratory Science: Christian Gram 1884
(http://www.hoslink.com/history2.htm#gram) . Hoslink. http://www.hoslink.com/history2.htm#gram. Retrieved
2010-07-27
4. ^ Beveridge TJ (2001). "Use of the Gram stain in microbiology". Biotech Histochem 76 (3): 111–8.
doi:10.1080/714028139 (http://dx.doi.org/10.1080%2F714028139) . PMID 11475313
(http://www.ncbi.nlm.nih.gov/pubmed/11475313) .
5. ^ a b Ryan KJ, Ray CG (editors) (2004). Sherris Medical Microbiology (4th ed.). McGraw Hill. pp. 232–3.
ISBN 0838585299.
6. ^ Madigan, MT; Martinko J, Parker J (2004). Brock Biology of Microorganisms (10th ed.). Lippincott Williams &
Wilkins. ISBN 0-13-066271-2.
7. ^ Søgaard M, Nørgaard M, Schønheyder H (2007). "First notification of positive blood cultures: high accuracy of
the Gram stain report (Epub ahead of publication)" (http://www.pubmedcentral.nih.gov/articlerender.fcgi?
tool=pmcentrez&artid=1865800) . J Clin Microbiol 45 (4): 1113. doi:10.1128/JCM.02523-06
(http://dx.doi.org/10.1128%2FJCM.02523-06) . PMID 17301283
(http://www.ncbi.nlm.nih.gov/pubmed/17301283) .
8. ^ Microbiology; J.G. Black Prentice Hall, 1993
9. ^ http://www.med-chem.com/procedures/GRAMSTAIN.pdf
10. ^ Llewellyn, Brian D. (May, 2005). "StainsFile - Stain theory - What a mordant is not"
(http://stainsfile.info/StainsFile/theory/notmrdnt.htm/) . http://stainsfile.info/StainsFile/theory/notmrdnt.htm/.
Retrieved 2009-09-10.
11. ^ Beveridge TJ, Davies JA (November 1983). "Cellular responses of Bacillus subtilis and Escherichia coli to the
Gram stain" (http://jb.asm.org/cgi/pmidlookup?view=long&pmid=6195148) . Journal of bacteriology 156 (2): 846–
58. PMID 6195148 (http://www.ncbi.nlm.nih.gov/pubmed/6195148) . PMC 217903
(http://www.pubmedcentral.gov/articlerender.fcgi?tool=pmcentrez&artid=217903) .
http://jb.asm.org/cgi/pmidlookup?view=long&pmid=6195148.
12. ^ Davies JA, Anderson GK, Beveridge TJ, Clark HC (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" (http://jb.asm.org/cgi/pmidlookup?view=long&pmid=6195147) . Journal of bacteriology 156 (2): 837–45.
PMID 6195147 (http://www.ncbi.nlm.nih.gov/pubmed/6195147) . PMC 217902
(http://www.pubmedcentral.gov/articlerender.fcgi?tool=pmcentrez&artid=217902) .
http://jb.asm.org/cgi/pmidlookup?view=long&pmid=6195147.
13. ^ Beveridge TJ (March 1990). "Mechanism of gram variability in select bacteria"
(http://jb.asm.org/cgi/pmidlookup?view=long&pmid=1689718) . Journal of bacteriology 172 (3): 1609–20.
PMID 1689718 (http://www.ncbi.nlm.nih.gov/pubmed/1689718) . PMC 208639
(http://www.pubmedcentral.gov/articlerender.fcgi?tool=pmcentrez&artid=208639) .
http://jb.asm.org/cgi/pmidlookup?view=long&pmid=1689718.

External links
Gram staining technique video (http://www.tgw1916.net/movies.html)
Gram staining procedure (http://www.microbeid.com/Methods/gramstain.html)
Retrieved from "http://en.wikipedia.org/wiki/Gram_staining"
en.wikipedia.org/wiki/Gram_staining 4/5
23-Oct-10 Gram staining - Wikipedia, the free e…
Categories: Bacteriology | Staining | Microbiology

This page was last modified on 19 October 2010 at 09:28.

Text is available under the Creative Commons Attribution-ShareAlike License; additional terms may apply.
See Terms of Use for details.
Wikipedia® is a registered trademark of the Wikimedia Foundation, Inc., a non-profit organization.

en.wikipedia.org/wiki/Gram_staining 5/5