Editing Gram stain (section)

== Staining mechanism ==
[[File:Gram Staining Bacteria.jpg|thumb|Purple-stained [[gram-positive]] (left) and pink-stained [[gram-negative]] (right)]]
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 do not rinse out during the staining procedure.
# The addition of [[iodine]], which binds to crystal violet and traps it in the cell
# Rapid decolorization with [[ethanol]] or [[acetone]]
# [[Counterstain]]ing with [[safranin]].<ref>{{cite book |title=Microbiology: Principles and Explorations |page=65 |first=Jacquelyn G. |last=Black |publisher=Prentice Hall |date=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 |work=Med-Chem.com |access-date=9 March 2016}}</ref>

{| class="wikitable"
|+Summary of Gram stain
! rowspan=2|Application of || rowspan=2|Reagent || colspan=4|Cell color
|-
! Gram-positive || Gram-negative
|-
| Primary dye || [[crystal violet]] || purple || purple
|-
| Trapper || [[iodine]] || purple || purple
|-
| Decolorizer || [[ethanol|alcohol]]/[[acetone]] || purple || colorless
|-
| Counter stain || [[safranin]]/[[carbol fuchsin]] || purple || pink or red
|}

[[Crystal violet]] (CV) dissociates in aqueous solutions into {{chem|CV|+}} and chloride ({{chem|Cl|-}}) ions. These ions penetrate the cell wall 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 |last=Leboffe |first=Michael |title=Microbiology Laboratory Theory and Application |date=2014 |publisher=Morton Publishing Company |location=Englewood, Colorado |isbn=978-1617312809 |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, colors the cell.<ref name="Stainsfile - Trapping Agents">{{cite web |url= http://stainsfile.info/StainsFile/theory/notmrdnt.htm |title=Stain theory – What a mordant is not |work=StainsFile.info |access-date=9 March 2016 |archive-url= https://web.archive.org/web/20160309162426/http://stainsfile.info/StainsFile/theory/notmrdnt.htm |archive-date=9 March 2016 |url-status=dead}}</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=Gram Stain |work=Microbugz |publisher=Austin Community College |access-date=2017-05-26 |archive-date=2017-05-26 |archive-url=https://web.archive.org/web/20170526012838/http://www.austincc.edu/microbugz/gram_stain.php |url-status=dead }}</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 |title=Pharmaceutical Microbiology: Essentials for Quality Assurance and Quality Control |last=Tim |first=Sandle |isbn=9780081000229 |oclc=923807961 |date=21 October 2015|publisher=Elsevier Science }}</ref> 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" /> 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">{{cite web |title=Gram's Serendipitous Stain |work=Hardy's Diagnostics |url=http://hardydiagnostics.com/wp-content/uploads/2016/05/Hans-Christian-Gram.pdf |archive-url=https://web.archive.org/web/20170324063915/http://hardydiagnostics.com/wp-content/uploads/2016/05/Hans-Christian-Gram.pdf |archive-date=2017-03-24 |url-status=live |last1=Hardy |first1=Jay |last2=Maria |first2=Santa}}</ref>

After decolorization, the gram-positive cell remains purple and the gram-negative cell loses its purple color.<ref name=":1" /> 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 |last1=Beveridge |first1=T. J. |last2=Davies |first2=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=10.1128/JB.156.2.846-858.1983}}</ref><ref name="Davies_et_al_1983">{{cite journal |last1=Davies |first1=J. A. |last2=Anderson |first2=G. K. |last3=Beveridge |first3=T. J. |last4=Clark |first4=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=10.1128/JB.156.2.837-845.1983}}</ref> 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.{{cn|date=May 2024}}