Examine individual changes

'{{short description|Method of staining used to differentiate bacterial species into two large groups (gram-positive and gram-negative)}} [[File:Gram stain 01.jpg|thumb|A Gram stain of mixed ''[[Staphylococcus aureus]]'' (''S. aureus'' ATCC 25923, gram-positive cocci, in purple) and ''[[Escherichia coli]]'' (''E. coli'' ATCC 11775, gram-negative bacilli, in red), the most common Gram stain reference bacteria]] '''Gram stain''' or '''Gram staining''', also called '''Gram's method''', is a method of [[staining]] used to distinguish and classify [[bacteria]]l species into two large groups ([[Gram-positive bacteria|gram-positive]] and [[Gram-negative bacteria|gram-negative]]). The name comes from the Danish [[bacteriologist]] [[Hans Christian Gram]], who developed the technique. Gram staining differentiates bacteria by the chemical and physical properties of their [[cell wall]]s 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.<ref name=Bergey_1994>{{cite book|author1=John G. Holt|author2=Noel R. Krieg|author3=Peter H.A. Sneath|author4=James T. Staley|author5=Stanley T. Williams|title=Bergey's Manual of Determinative Bacteriology|edition=9th|publisher=Lippincott Williams & Wilkins|year=1994|page=11|isbn=0-683-00603-7}}</ref> Gram-positive bacteria retain the [[Crystal violet|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 unseen on Gram-positive bacteria because of the darker crystal violet stain. The 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. ==History== The method is named after its inventor, the [[Denmark|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]] 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.<ref>{{Cite journal|last=Austrian|first=R.|year=1960|title=The Gram stain and the etiology of lobar pneumonia, an historical note|journal=Bacteriological Reviews|volume=24|issue=3|pages=261–265|pmid=13685217|pmc=441053}}</ref> He published his method in 1884, and included in his short report the observation that the [[typhus]] [[bacillus (shape)|bacillus]] did not retain the stain.<ref name=Gram_1884>{{Cite journal |authorlink = Hans Christian Gram |last = Gram |first = H.C. |title = Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten |journal = Fortschritte der Medizin |language=German |year = 1884 |volume = 2 |pages = 185–189}}.<br>English translation in: {{Cite book|last=Brock|first=T.D.|year=1999|title=Milestones in Microbiology 1546–1940|edition=2|publisher=ASM Press|place= |pages=215–218|isbn=1-55581-142-6|url=https://books.google.com/?id=q5JHcs8w21gC&lpg=PP1&dq=Milestones%20in%20Microbiology&pg=PA215#v=onepage&q}}<br/>Translation is also at: {{Cite journal|url=http://www.hoslink.com/pathology/lab_general/history2.htm#gram|last=Brock|first=T.D.|title=Pioneers in Medical Laboratory Science: Christian Gram 1884|publisher=Hoslink|accessdate=2010-07-27}}</ref> ==Uses== Gram staining is a [[bacteriology|bacteriological]] [[laboratory]] technique<ref name=Sherris>{{cite book |author = Ryan K.J., Ray C.G. (editors) |title = Sherris Medical Microbiology |pages = 232f |edition = 4th |publisher = McGraw Hill |year = 2004 |isbn= 0838585299 }}{{full citation needed|date=March 2016}}</ref> used to differentiate [[bacterium|bacterial]] species into two large groups ([[gram-positive]] and [[gram-negative]]) based on the physical properties of their [[cell wall]]s.<ref name=Madigan_2004>{{cite book |last = Madigan |first = M.T. |author2=Martinko J. |author3=Parker J. |title = Brock Biology of Microorganisms |edition = 10th |publisher = Lippincott Williams & Wilkins |year = 2004 |isbn = 0-13-066271-2 }}{{full citation needed|date=March 2016}}</ref>{{page needed|date=March 2016}} Gram staining is not used to classify [[archaea]], formerly archaeabacteria, since these microorganisms yield widely varying responses that do not follow their [[phylogenetics|phylogenetic groups]].<ref>{{cite journal |author=Beveridge T.J. |title=Use of the Gram stain in microbiology |journal=Biotechnic & Histochemistry |volume=76 |issue=3 |pages=111–118 |year=2001 |pmid=11475313 |doi=10.1080/714028139}}</ref> The 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 [[polymerase chain reaction|PCR]] in one primary research report regarding gonococcal urethritis.<ref>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 Gram stain for diagnosis of gonococcal urethritis. Indian Journal of Dermatology, Venereology, and Leprology, (2009); 75, 101.{{primary source inline|date=March 2016}}</ref>{{primary source inline|date=March 2016}} ===Medical=== {{see also|Gram-negative bacterial infection|Gram-positive bacterial infection}} Gram stains are performed on [[body fluid]] or [[biopsy]] when [[infection]] is suspected. Gram stains yield results much more quickly than [[microbiological culture|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 arthritis]].<ref name=Sherris /><ref name="Søgaard_2007">{{cite journal |author1=Søgaard M. |author2=Nørgaard M. |author3=Schønheyder H. |title=First notification of positive blood cultures: high accuracy of the Gram stain report |journal=Journal of Clinical Microbiology |volume= 45|issue= 4|pages= 1113–1117|year= 2007 |pmid=17301283 |doi=10.1128/JCM.02523-06 |pmc=1865800}}</ref> ===Staining mechanism=== 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 ([[gentian violet|crystal violet]]) to a heat-fixed smear of a bacterial culture. [[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 the cell *Rapid decolorization with [[ethanol]] or [[acetone]] *''[[Counterstain]]ing'' with [[safranin]].<ref>Microbiology: Principles and Explorations, p 65; Jacquelyn.G. Black Prentice Hall, 1993.</ref> [[Carbol fuchsin]] is sometimes substituted for safranin since it more intensely stains anaerobic bacteria, but it is less commonly used as a counterstain.<ref>{{cite web|url=http://www.med-chem.com/index.php?url=lab_procedures&subsection=gram_staining|title=Medical Chemical Corporation|date=|work=med-chem.com|accessdate=9 March 2016}}</ref> [[File:Gram Stain Procedure.png|thumb|Figure 1: Gram stain procedure and effect on Gram-negative and Gram-positive cell walls.]] [[Crystal violet]] (CV) dissociates in aqueous solutions into {{chem|CV|+}} and chloride ({{chem|Cl|-}}) ions. These ions penetrate through the cell wall and cell membrane of both gram-positive and gram-negative cells. The {{chem|CV|+}} ion interacts with negatively charged components of bacterial cells and stains the cells purple.<ref>{{cite book|last1=Leboffe|first1=Michael|title=Microbiology Laboratory Theory and Application|date=2014|publisher=Morton Publishing Company|location=Englewood, CO|isbn=1617312800|pages=105|edition=3rd}}</ref> Iodide ({{chem|I|-}} or {{chem|I|3|-}}) interacts with {{chem|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.<ref name="Stainsfile - Trapping Agents">{{cite web|url=http://stainsfile.info/StainsFile/theory/notmrdnt.htm|title=StainsFile - Stain theory - What a mordant is not|date=|work=stainsfile.info|accessdate=9 March 2016}}</ref> When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane.<ref>{{Cite web|url=http://www.austincc.edu/microbugz/gram_stain.php|title=Welcome to Microbugz - Gram Stain|website=www.austincc.edu|access-date=2017-05-26}}</ref> 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.<ref name=":0">{{Cite book|url=https://www.worldcat.org/oclc/923807961|title=Pharmaceutical microbiology : essentials for quality assurance and quality control|last=Tim,|first=Sandle,|isbn=9780081000229|oclc=923807961}}</ref>{{citation needed|date=March 2016}} 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.<ref name=":0" />{{citation needed|date=March 2016}} 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).<ref name=":1">Gram's Serendipitous Stain by Hardy's Diagnostics at hardydiagnostics.com/wp-content/uploads/2016/05/Hans-Christian-Gram.pdf</ref>{{citation needed|date=March 2016}} After decolorization, the gram-positive cell remains purple and the gram-negative cell loses its purple color.<ref name=":1" />{{citation needed|date=March 2016}} Counterstain, which is usually positively charged safranin or basic fuchsine, is applied last to give decolorized gram-negative bacteria a pink or red color.<ref name="Beveridge_and_Davies_1983">{{cite journal |author1=Beveridge T.J. |author2=Davies J.A. |title=Cellular responses of Bacillus subtilis and Escherichia coli to the Gram stain |journal=Journal of Bacteriology |volume=156 |issue=2 |pages=846–58 |date=November 1983 |pmid=6195148 |pmc=217903 |doi= |url=http://jb.asm.org/cgi/pmidlookup?view=long&pmid=6195148}}</ref><ref name="Davies_et_al_1983">{{cite journal |author1=Davies J.A. |author2=Anderson G.K. |author3=Beveridge T.J. |author4=Clark H.C. |title=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=Journal of Bacteriology |volume=156 |issue=2 |pages=837–845 |date=November 1983 |pmid=6195147 |pmc=217902 |doi= |url=http://jb.asm.org/cgi/pmidlookup?view=long&pmid=6195147}}</ref> ==Examples== ===Gram-positive bacteria=== {{Main article|Gram-positive bacteria}} Gram-positive bacteria generally have a single membrane (''monoderm'') surrounded by a thick peptidoglycan. This rule is followed by two phyla: ''[[Firmicutes]]'' (except for the classes [[Mollicutes]] and [[Negativicutes]]) and the ''[[Actinobacteria]]''.<ref name="Madigan_2004" /><ref name="Begey_essay">{{cite book |series=Bergey's Manual of Systematic Bacteriology|volume=2A|title=Introductory Essays|editor=George M. Garrity |authors=Don J. Brenner, Noel R. Krieg, James T. Staley |publisher=Springer|location= New York |edition=2nd|isbn=978-0-387-24143-2 |pages=304 |url=https://www.springer.com/life+sciences/book/978-0-387-24143-2 |date=July 26, 2005 |origyear=1984|id=British Library no. GBA561951}}{{Page needed|date=October 2013}}</ref> In contrast, members of the [[Chloroflexi (phylum)|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|Deinococcus-Thermus group]], stain positive but are diderms with a thick peptidoglycan.<ref name="Madigan_2004" />{{page needed|date=March 2016}} <ref name="Begey_essay" /> [[Bacterial taxonomy|Historically]], the gram-positive forms made up the [[phylum (biology)|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]]''.<ref>{{Cite journal|last=Galperin|first=Michael Y.|date=2013-12-27|title=Genome Diversity of Spore-Forming Firmicutes|url=http://www.asmscience.org/content/journal/microbiolspec/10.1128/microbiolspectrum.TBS-0015-2012|journal=Microbiology Spectrum|language=en|volume=1|issue=2|doi=10.1128/microbiolspectrum.tbs-0015-2012|issn=2165-0497|pmc=4306282|pmid=26184964}}</ref>{{citation needed|date=March 2016}} 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.<ref>Practical Medical Microbiology by Dr. Hams H. Hashem, from http://qu.edu.iq/el/mod/resource/view.php?id=1391</ref>{{citation needed|date=March 2016}} 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-fastness|acid fast bacteria]], and can only be differentiated from other gram-positive bacteria by [[Ziehl–Neelsen stain|special staining procedures]].<ref>{{Cite web|url=http://www2.highlands.edu/academics/divisions/scipe/biology/labs/rome/acid_fast_stain.htm|title=THE ACID FAST STAIN|website=www2.highlands.edu|access-date=2017-06-09}}</ref>{{citation needed|date=March 2016}} ===Gram-negative bacteria=== {{Main article|Gram-negative bacteria}} <!--Definition of Gr- purposefully repeated-->Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes (''diderms''). Most [[bacterial phyla]] are gram-negative, including the [[cyanobacteria]], [[green sulfur bacteria]], and most [[Proteobacteria]] (exceptions being some members of the ''[[Rickettsiales]]'' and the insect-endosymbionts of the ''[[Enterobacteriales]]'').<ref name="Madigan_2004" />{{page needed|date=March 2016}}<ref name="Begey_essay" /> ===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.<ref name=":0" />{{citation needed|date=October 2015}} 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.<ref name="Beveridge_1990">{{cite journal |last=Beveridge|first=Terry J.|title=Mechanism of gram variability in select bacteria |journal=Journal of Bacteriology |volume=172 |issue=3 |pages=1609–20 |date=March 1990 |pmid=1689718 |pmc=208639 |doi= 10.1128/jb.172.3.1609-1620.1990|url=http://jb.asm.org/cgi/pmidlookup?view=long&pmid=1689718}}</ref> In addition, in all bacteria stained using the Gram stain, the age of the culture may influence the results of the stain.<ref name="beveridge">{{cite journal|last=Beveridge|first=Terry J.|title=Mechanism of Gram Variability in Select Bacteria.|journal=Journal of Bacteriology|date=March 1990|volume=172|issue=3|pages=1609–20|pmid=1689718|url=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC208639/pdf/jbacter01045-0455.pdf|accessdate=17 September 2016|doi=10.1128/jb.172.3.1609-1620.1990|pmc=208639}}</ref> 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 ''[[Mycobacterium tuberculosis|M. tuberculosis]]'' and ''[[Mycobacterium leprae|M. leprae]]''.<ref>{{cite book|last=Black|first=Jacquelyn|title=Microbiology: Principles and Exploration|edition=8th|date=2012|publisher=John Wiley & Sons|isbn=978-0-470-54109-8|page=68}}</ref><ref name="pmid19885935">{{cite journal |author1=Reynolds J. |author2=Moyes R.B. |author3=Breakwell D.P. |title=Differential staining of bacteria: acid fast stain|journal=[[Current Protocols#Titles|Current Protocols in Microbiology]]|year=2009|pages=Appendix 3H | doi=10.1002/9780471729259.mca03hs15|pmid=19885935|volume=Appendix 3}}</ref> == Orthographic note == 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.<ref>{{cite book |url=https://books.google.com/books?id=btb1AwAAQBAJ&pg=PA105 |title=New Hart's Rules: The Oxford Style Guide|first= Anne |last=Waddingham |page=105 |publisher=OUP Oxford |date=28 August 2014 |isbn= 978-0199570027 }}</ref> The adjectives 'gram-positive' and 'gram-negative'; as [[eponym#Orthographic conventions|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]].<ref>Centers for Disease Control and Prevention. Emerging Infectious Diseases Journal Style Guide. [http://wwwnc.cdc.gov/eid/pages/preferred-usage.htm Preferred Usage]</ref> Dictionaries may use lowercase,<ref name="Dorlands">{{Citation |author=Elsevier |authorlink=Elsevier |title=Dorland's Illustrated Medical Dictionary |publisher=Elsevier |url=http://www.dorlands.com/ |postscript=.}}</ref><ref name="MWMD">{{Citation |author=Merriam-Webster |authorlink=Merriam-Webster |title=gram–positive |publisher=Merriam-Webster |url=http://www.merriam-webster.com/dictionary/gram%E2%80%93positive |postscript=.}}</ref> uppercase,<ref>{{cite web |url=http://www.collinsdictionary.com/dictionary/english/gram-positive |title=Definition of Gram-positive |work=Collins}}</ref><ref>{{cite web |url= https://en.oxforddictionaries.com/definition/gram_stain |title=Gram stain |work=Oxford Dictionary}}</ref><ref>{{cite web |url=http://www.medicinenet.com/script/main/art.asp?articlekey=9585 |title=Definition of Gram-positive |work=Medicinenet}}</ref><ref>{{cite web |url=http://www.businessdictionary.com/definition/Gram-negative-positive.html |title=Gram negative/positive |work= Business dictionary}}</ref> or both.<ref>{{cite web |url=https://ahdictionary.com/word/search.html?q=gram+positive&submit.x=0&submit.y=0 |title=gram-pos·i·tive or Gram-pos·i·tive |work=The American Heritage Dictionary }}</ref><ref name="dictionary">{{cite web|url=http://www.dictionary.com/browse/gram-positive |title=Gram-positive |work=Dictionary.com}}</ref> Uppercase 'Gram-positive' or 'Gram-negative' usage is also common in many [[scientific journal]] articles and publications.<ref name="dictionary"/><ref>{{cite journal |url=http://www.nature.com/nrmicro/journal/v13/n10/full/nrmicro3480.html |title=Through the wall: extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi |authors=Lisa Brown, Julie M. Wolf, Rafael Prados-Rosales & Arturo Casadevall|journal=Nature Reviews Microbiology |volume=13 |pages= 620–630 |date=2015 |doi= 10.1038/nrmicro3480 |pmid=26324094 |pmc=4860279}}</ref><ref>{{cite journal |journal=Science |url=http://science.sciencemag.org/content/348/6240/1218.15 |title=Detecting Gram-negative bacteria |date=12 June 2015 |volume=348|issue= 6240|page= 1218|doi= 10.1126/science.348.6240.1218-o |author= Kristen L. Mueller}}</ref> When articles are submitted to journals, each journal may or may not apply house style to the [[postprint]] version. [[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 stain'. ==See also== *[[Bacterial cell structure]] ==References== {{Reflist}} ==External links== {{Commons category|Gram stains}} {{Wikibooks|School Science|Gram staining}} *[http://www.tgw1916.net/movies.html Gram staining technique video] {{Stains}} [[Category:Articles with inconsistent citation formats]] [[Category:Bacteriology]] [[Category:Staining]] [[Category:Microscopy]] [[Category:Danish inventions]] [[Category:1884 in biology]]'