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Gram stain

Gram stain or Gram staining, also called Gram'smethod, is a method of staining used to distinguish andclassify bacterial species into two large groups (Gram-positive and Gram-negative). The name comes from

A Gram stain of mixed Staphylococcus aureus (S. aureus ATCC 25923,Gram-positive cocci, in purple) and Escherichia coli (E. coli ATCC11775, Gram-negative bacilli, in red), the most common Gram stainreference bacteria

the Danish bacteriologist Hans Christian Gram, whodeveloped the technique.[1]

Gram staining differentiates bacteria by the chemicaland physical properties of their cell walls. Gram-positive cells have a thick layer of peptidoglycan in thecell wall that retains the primary stain, crystal violet.Gram-negative cells have a thinner peptidoglycan layerthat allows the crystal violet to wash out. They arestained pink or red by the counterstain,[2] commonlysafranin or fuchsine.

The Gram stain is almost always the first step in thepreliminary identification of a bacterial organism. WhileGram staining is a valuable diagnostic tool in bothclinical and research settings, not all bacteria can bedefinitively classified by this technique. This gives riseto Gram-variable and Gram-indeterminate groups.

History

The method is named after its inventor, the Danishscientist Hans Christian Gram (1853–1938), whodeveloped the technique while working with CarlFriedländer in the morgue of the city hospital in Berlin in1884. Gram devised his technique not for the purposeof distinguishing one type of bacterium from anotherbut to make bacteria more visible in stained sections oflung tissue.[3] He published his method in 1884, andincluded in his short report the observation that thetyphus bacillus did not retain the stain.[4]

Gram staining is a bacteriological laboratorytechnique[5] used to differentiate bacterial species intotwo large groups (Gram-positive and Gram-negative)based on the physical properties of their cell walls.[6]

Gram staining is not used to classify archaea, formerlyarchaeabacteria, since these microorganisms yield

Uses

widely varying responses that do not follow theirphylogenetic groups.[7]

The Gram stain is not an infallible tool for diagnosis,identification, or phylogeny, and it is of extremelylimited use in environmental microbiology. It is usedmainly to make a preliminary morphologic identificationor to establish that there are significant numbers ofbacteria in a clinical specimen. It cannot identifybacteria to the species level, and for most medicalconditions, it should not be used as the sole method ofbacterial identification. In clinical microbiologylaboratories, it is used in combination with othertraditional and molecular techniques to identifybacteria. Some organisms are Gram-variable (meaningthey may stain either negative or positive); some arenot stained with either dye used in the Gram techniqueand are not seen. In a modern environmental ormolecular microbiology lab, most identification is doneusing genetic sequences and other molecular

techniques, which are far more specific and informativethan differential staining.

Gram staining has been suggested to be as effective adiagnostic tool as PCR in one primary research reportregarding gonococcal urethritis.[8]

Medical

Gram stains are performed on body fluid or biopsywhen infection is suspected. Gram stains yield resultsmuch more quickly than culturing, and is especiallyimportant when infection would make an importantdifference in the patient's treatment and prognosis;examples are cerebrospinal fluid for meningitis andsynovial fluid for septic arthritis.[5][9]

Staining mechanism

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Gram-positive bacteria have a thick mesh-like cell wallmade of peptidoglycan (50–90% of cell envelope), andas a result are stained purple by crystal violet, whereasGram-negative bacteria have a thinner layer (10% of cellenvelope), so do not retain the purple stain and arecounter-stained pink by safranin. There are four basicsteps of the Gram stain:

Applying a primary stain (crystal violet) to a heat-fixed smear of a bacterial culture. Heat fixation killssome bacteria but is mostly used to affix the bacteriato the slide so that they don't rinse out during thestaining procedure.

Purple-stained Gram-positive (left) and pink-stained Gram-negative(right)

The addition of iodide, which binds to crystal violetand traps it in the cell

Rapid decolorization with ethanol or acetone

Counterstaining with safranin.[10] Carbol fuchsin issometimes substituted for safranin since it moreintensely stains anaerobic bacteria, but it is lesscommonly used as a counterstain.[11]

Summary of Gram stain

Application of ReagentCell color

Gram-positive Gram-negative

Primary dye crystal violet purple purple

Trapping agent iodine purple purple

Decolorizer alcohol/acetone purple colorless

Counter stain safranin/carbol fuchsin purple pink

Crystal violet (CV) dissociates in aqueous solutions

into CV+ and chloride (Cl−) ions. These ions penetratethe cell wall of both Gram-positive and Gram-negative

cells. The CV+ ion interacts with negatively chargedcomponents of bacterial cells and stains the cellspurple.[12]

Iodide (I− or I−3) interacts with CV+ and forms largecomplexes of crystal violet and iodine (CV–I) within theinner and outer layers of the cell. Iodine is oftenreferred to as a mordant, but is a trapping agent thatprevents the removal of the CV–I complex and,therefore, colors the cell.[13]

When a decolorizer such as alcohol or acetone isadded, it interacts with the lipids of the cellmembrane.[14] A Gram-negative cell loses its outerlipopolysaccharide membrane, and the innerpeptidoglycan layer is left exposed. The CV–Icomplexes are washed from the gram-negative cellalong with the outer membrane.[15] In contrast, a Gram-positive cell becomes dehydrated from an ethanoltreatment. The large CV–I complexes become trappedwithin the Gram-positive cell due to the multilayerednature of its peptidoglycan.[15] The decolorization stepis critical and must be timed correctly; the crystal violetstain is removed from both Gram-positive and negative

cells if the decolorizing agent is left on too long (amatter of seconds).[16]

After decolorization, the Gram-positive cell remainspurple and the Gram-negative cell loses its purplecolor.[16] Counterstain, which is usually positivelycharged safranin or basic fuchsine, is applied last togive decolorized Gram-negative bacteria a pink or redcolor.[2][17] Both Gram-positive bacteria and Gram-negative bacteria pick up the counterstain. Thecounterstain, however, is unseen on Gram-positivebacteria because of the darker crystal violet stain.

Gram-positive bacteria

Gram-positive bacteria generally have a singlemembrane (monoderm) surrounded by a thickpeptidoglycan. This rule is followed by two phyla:

Examples

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Firmicutes (except for the classes Mollicutes andNegativicutes) and the Actinobacteria.[6][18] In contrast,members of the Chloroflexi (green non-sulfur bacteria)are monoderms but possess a thin or absent (classDehalococcoidetes) peptidoglycan and can stainnegative, positive or indeterminate; members of theDeinococcus–Thermus group stain positive but arediderms with a thick peptidoglycan.[6][18]

Historically, the Gram-positive forms made up thephylum Firmicutes, a name now used for the largestgroup. It includes many well-known genera such asLactobacillus, Bacillus, Listeria, Staphylococcus,Streptococcus, Enterococcus, and Clostridium.[19] It hasalso been expanded to include the Mollicutes, bacteriasuch as Mycoplasma and Thermoplasma that lack cellwalls and so cannot be Gram-stained, but are derivedfrom such forms.[20]

Some bacteria have cell walls which are particularlyadept at retaining stains. These will appear positive byGram stain even though they are not closely related toother Gram-positive bacteria. These are called acid-fastbacteria, and can only be differentiated from otherGram-positive bacteria by special stainingprocedures.[21]

Gram-negative bacteria

Gram-negative bacteria generally possess a thin layerof peptidoglycan between two membranes (diderms).Most bacterial phyla are Gram-negative, including thecyanobacteria, green sulfur bacteria, and mostProteobacteria (exceptions being some members ofthe Rickettsiales and the insect-endosymbionts of theEnterobacteriales).[6][18]

Gram-variable and Gram-indeterminate

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bacteria

Some bacteria, after staining with the Gram stain, yielda Gram-variable pattern: a mix of pink and purple cellsare seen.[15][22] In cultures of Bacillus, Butyrivibrio, andClostridium, a decrease in peptidoglycan thicknessduring growth coincides with an increase in the numberof cells that stain Gram-negative.[23] In addition, in allbacteria stained using the Gram stain, the age of theculture may influence the results of the stain.[23]

Gram-indeterminate bacteria do not respondpredictably to Gram staining and, therefore, cannot bedetermined as either Gram-positive or Gram-negative.Examples include many species of Mycobacterium,including Mycobacterium bovis, Mycobactrium lepraeand Mycobacterium tuberculosis, the latter two of whichare the causative agents of leprosy and tuberculosisrespectively.[24][25]

The term "Gram staining" is derived from the surnameof Hans Christian Gram; the eponym (Gram) istherefore capitalized but not the common noun (stain)as is usual for scientific terms.[26] The initial letters of"Gram-positive" and "Gram-negative", which areeponymous adjectives, can be either lowercase "g" orcapital "G", depending on what style guide (if any)governs the document being written. Lowercase style isused by the US Centers for Disease Control andPrevention and other style regimens such as the AMAstyle.[27] Dictionaries may use lowercase,[28][29]

uppercase,[30][31][32][33] or both.[34][35] Uppercase 'Gram-positive' or 'Gram-negative' usage is also common inmany scientific journal articles andpublications.[35][36][37] When articles are submitted tojournals, each journal may or may not apply house styleto the postprint version. Preprint versions containwhichever style the author happened to use. Even style

Orthographic note

regimens that use lowercase for the adjectives "gram-positive" and "gram-negative" still use capital for "Gramstain".

Bacterial cell structure

Ziehl–Neelsen stain

1. Colco, R (2005). "Gram Staining". Current Protocolsin Microbiology. 00 (1): Appendix 3C.doi:10.1002/9780471729259.mca03cs00 .ISBN 978-0471729259. PMID 18770544 .

2. Beveridge T. J.; Davies J. A. (November 1983)."Cellular responses of Bacillus subtilis andEscherichia coli to the Gram stain" . Journal ofBacteriology. 156 (2): 846–58.doi:10.1128/JB.156.2.846-858.1983 .PMC 217903 . PMID 6195148 .

See also

References

3. Austrian, R. (1960). "The Gram stain and theetiology of lobar pneumonia, an historical note" .Bacteriological Reviews. 24 (3): 261–265.doi:10.1128/MMBR.24.3.261-265.1960 .PMC 441053 . PMID 13685217 .

4. Gram, H.C. (1884). "Über die isolierte Färbung derSchizomyceten in Schnitt- und Trockenpräparaten".Fortschritte der Medizin (in German). 2: 185–189..English translation in: Brock, T.D. (1999). Milestonesin Microbiology 1546–1940 (2 ed.). ASM Press.pp. 215–218. ISBN 978-1-55581-142-6.Translation is also at: Brock, T.D. "Pioneers inMedical Laboratory Science: Christian Gram 1884" .Hoslink. Retrieved 2010-07-27.

5. Ryan K.J., Ray C.G. (editors) (2004). Sherris MedicalMicrobiology (4th ed.). McGraw Hill. pp. 232f.ISBN 978-0838585290.

6. Madigan, M.T.; Martinko J.; Parker J. (2004). BrockBiology of Microorganisms (10th ed.). LippincottWilliams & Wilkins. ISBN 978-0-13-066271-2.

7. Beveridge T.J. (2001). "Use of the Gram stain inmicrobiology". Biotechnic & Histochemistry. 76 (3):111–118. doi:10.1080/714028139 .PMID 11475313 .

8. El-Garnal, A.H., Al-Otaibi, S.R., Alshamali, A.,Abdulrazzaq, A., Najem, N., and Fouzan, A.A.Polymerase chain reaction is no better than Gramstain for diagnosis of gonococcal urethritis. IndianJournal of Dermatology, Venereology and Leprology,(2009); 75, 101.

9. Søgaard M.; Nørgaard M.; Schønheyder H. (2007)."First notification of positive blood cultures: highaccuracy of the Gram stain report" . Journal ofClinical Microbiology. 45 (4): 1113–1117.doi:10.1128/JCM.02523-06 . PMC 1865800 .PMID 17301283 .

10. Microbiology: Principles and Explorations, p 65;Jacquelyn G. Black, Prentice Hall, 1993.

11. "Medical Chemical Corporation" . med-chem.com.Retrieved 9 March 2016.

12. Leboffe, Michael (2014). Microbiology LaboratoryTheory and Application (3rd ed.). Englewood, CO:Morton Publishing Company. p. 105. ISBN 978-1617312809.

13. "StainsFile - Stain theory - What a mordant is not" .stainsfile.info. Retrieved 9 March 2016.

14. "Welcome to Microbugz - Gram Stain" .www.austincc.edu. Retrieved 2017-05-26.

15. Tim, Sandle (2015-10-21). Pharmaceuticalmicrobiology : essentials for quality assurance andquality control. ISBN 9780081000229.OCLC 923807961 .

16. Hardy, Jay; Maria, Santa. "Gram's SerendipitousStain" (PDF). Hardy's Diagnostics.

17. Davies J. A.; Anderson G. K.; Beveridge T. J.; ClarkH. C. (November 1983). "Chemical mechanism ofthe Gram stain and synthesis of a new electron-opaque marker for electron microscopy, whichreplaces the iodine mordant of the stain" . Journalof Bacteriology. 156 (2): 837–845.doi:10.1128/JB.156.2.837-845.1983 .PMC 217902 . PMID 6195147 .

18. Don J. Brenner, Noel R. Krieg, James T. Staley (July26, 2005) [1984]. George M. Garrity (ed.).Introductory Essays . Bergey's Manual ofSystematic Bacteriology. 2A (2nd ed.). New York:Springer. p. 304. ISBN 978-0-387-24143-2. BritishLibrary no. GBA561951.

19. Galperin, Michael Y. (2013-12-27). "GenomeDiversity of Spore-Forming Firmicutes" .Microbiology Spectrum. 1 (2).doi:10.1128/microbiolspectrum.tbs-0015-2012 .ISSN 2165-0497 . PMC 4306282 .PMID 26184964 .

20. Practical Medical Microbiology by Hams H.Hashem, fromhttp://qu.edu.iq/el/mod/resource/view.php?id=1391

21. "The Acid Fast Stain" . www2.highlands.edu.Archived from the original on 2017-06-10.Retrieved 2017-06-09.

22. "Mechanism of Gram Variability in Select Bacteria"(PDF). Journal of Bacteriology. Retrieved2020-01-24.

23. Beveridge, Terry J. (March 1990). "Mechanism ofgram variability in select bacteria" . Journal ofBacteriology. 172 (3): 1609–20.doi:10.1128/jb.172.3.1609-1620.1990 .PMC 208639 . PMID 1689718 .

24. Black, Jacquelyn (2012). Microbiology: Principlesand Exploration (8th ed.). John Wiley & Sons. p. 68.ISBN 978-0-470-54109-8.

25. 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 .ISBN 978-0471729259. PMID 19885935 .

26. Waddingham, Anne (28 August 2014). New Hart'sRules: The Oxford Style Guide . OUP Oxford. p. 105.ISBN 978-0199570027.

27. Centers for Disease Control and Prevention.Emerging Infectious Diseases Journal Style Guide.Preferred Usage

28. Elsevier, Dorland's Illustrated Medical Dictionary ,Elsevier, archived from the original on 2014-01-11,retrieved 2016-10-20.

29. Merriam-Webster, gram–positive , Merriam-Webster.

30. "Definition of Gram-positive" . Collins.

31. "Gram stain" . Oxford Dictionary.

32. "Definition of Gram-positive" . Medicinenet.

33. "Gram negative/positive" . Business dictionary.

34. "gram-pos·i·tive or Gram-pos·i·tive" . The AmericanHeritage Dictionary.

35. "Gram-positive" . Dictionary.com.

Wikimedia Commons has media related to Gramstains.

The Wikibook School Science has a page on thetopic of: Gram staining

Gram staining technique video

36. Lisa Brown, Julie M. Wolf, Rafael Prados-Rosales &Arturo Casadevall (2015). "Through the wall:extracellular vesicles in Gram-positive bacteria,mycobacteria and fungi" . Nature ReviewsMicrobiology. 13 (10): 620–630.doi:10.1038/nrmicro3480 . PMC 4860279 .PMID 26324094 .

37. Kristen L. Mueller (12 June 2015). "Detecting Gram-negative bacteria". Science. 348 (6240): 1218.doi:10.1126/science.348.6240.1218-o .

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