gram stain

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 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.{{Cite journal |last=Austrian |first=R. |date=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 |doi=10.1128/MMBR.24.3.261-265.1960}} Gram noticed that some bacterial cells possessed noticeable resistance to decolorization. Based on these observations, Gram developed the initial gram staining procedure, initially making use of Ehrlich's aniline-gentian violet, Lugol's iodine, absolute alcohol for decolorization, and Bismarck brown for counterstain.{{Cite journal |last1=Bartholomew |first1=James W. |last2=Mittwer |first2=Tod |date=March 1952 |title=THE GRAM STAIN |journal=Bacteriological Reviews |language=en |volume=16 |issue=1 |pages=1–29 |doi=10.1128/br.16.1.1-29.1952 |issn=0005-3678 |pmc=180726 |pmid=14925025}} He published his method in 1884, and included in his short report the observation that the typhus bacillus did not retain the stain.{{Cite journal |author-link=Hans Christian Gram |last=Gram |first=Hans Christian |title=Über die isolierte Färbung der Schizomyceten in Schnitt- und Trockenpräparaten |journal=Fortschritte der Medizin |language=de |date=1884 |volume=2 |pages=185–189}}.
English translation in: {{Cite book |last=Brock |first=T. D. |date=1999 |title=Milestones in Microbiology 1546–1940 |edition=2nd |publisher=ASM Press |pages=215–218 |isbn=978-1-55581-142-6 |url=https://books.google.com/books?id=q5JHcs8w21gC&q=Milestones%20in%20Microbiology&pg=PA215 }}
Translation is also at: {{Cite web |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 |work=HOSLink.com |access-date=27 July 2010 |archive-date=10 August 2016 |archive-url=https://web.archive.org/web/20160810225816/http://www.hoslink.com/pathology/lab_general/history2.htm#gram |url-status=dead }} Gram did not initially make the distinction between Gram-negative and Gram-positive bacteria using his procedure.

File:Candida Gram stain.jpg from a vaginal swab. The small oval chlamydospores are 2–4 μm in diameter.]]

Gram staining is a bacteriological laboratory technique{{cite book |editor1-last=Ryan |editor1-first=K. J. |editor2-last=Ray |editor2-first=C. G. |title=Sherris Medical Microbiology |pages=232f |edition=4th |publisher=McGraw Hill |date=2004 |isbn=978-0838585290}} used to differentiate bacterial species into two large groups (gram-positive and gram-negative) based on the physical properties of their cell walls.{{cite book |last1=Madigan |first1=M. T. |last2=Martinko |first2=J. |last3=Parker |first3=J. |title=Brock Biology of Microorganisms |edition=10th |publisher=Lippincott Williams & Wilkins |date=2004 |isbn=978-0-13-066271-2 |url-access=registration |url= https://archive.org/details/brockbiologyofmi00madi}}{{page needed|date=March 2016}} Gram staining can also be used to diagnose a fungal infection. Gram staining is not used to classify archaea, since these microorganisms yield widely varying responses that do not follow their phylogenetic groups.{{cite journal |last=Beveridge |first=T. J. |title=Use of the Gram stain in microbiology |journal=Biotechnic & Histochemistry |volume=76 |issue=3 |pages=111–118 |date=2001 |pmid=11475313 |doi=10.1080/714028139}}

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.{{cn|date=May 2024}}

{{see also|Pathogenic bacteria}}

Gram stains are performed on body fluid or biopsy when infection is suspected. Gram stains yield results much more quickly than culturing, and are 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.{{cite journal |last1=Søgaard |first1=M. |last2=Nørgaard |first2=M. |last3=Schønheyder |first3=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 |date=2007 |pmid=17301283 |doi=10.1128/JCM.02523-06 |pmc=1865800}}

File:Gram Staining Bacteria.jpg (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:

1. Applying a primary stain (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.
2. The addition of iodine, which binds to crystal violet and traps it in the cell
3. Rapid decolorization with ethanol or acetone
4. Counterstaining with safranin.{{cite book |title=Microbiology: Principles and Explorations |page=65 |first=Jacquelyn G. |last=Black |publisher=Prentice Hall |date=1993}} Carbol fuchsin is sometimes substituted for safranin since it more intensely stains anaerobic bacteria, but it is less commonly used as a counterstain.{{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}}

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.{{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}}

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.{{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}}

When a decolorizer such as alcohol or acetone is added, it interacts with the lipids of the cell membrane.{{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 }} 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.{{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 }} 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).{{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}}

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.{{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}}{{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}} 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}}

File:Gram-positive bacteria and pus cells.jpg streptococci surrounded by pus cells]]

{{Main|Gram-positive bacteria}}

Gram-positive bacteria generally have a single membrane (monoderm) surrounded by a thick peptidoglycan.

This rule is followed by two phyla: Bacillota (except for the classes Mollicutes and Negativicutes) and the Actinomycetota.{{cite book |series=Bergey's Manual of Systematic Bacteriology |volume=2A |title=Introductory Essays |editor-first=George M. |editor-last=Garrity |first1=Don J. |last1=Brenner |first2=Noel R. |last2=Krieg |first3=James T. |last3=Staley |publisher=Springer |location= New York |edition=2nd |isbn=978-0-387-24143-2 |page=304 |url= https://www.springer.com/life+sciences/book/978-0-387-24143-2 |date=26 July 2005 |orig-year=1984 |id=British Library no. GBA561951}} In contrast, members of the Chloroflexota (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 Deinococcota stain positive but are diderms with a thick peptidoglycan.{{page needed|date=March 2016}}

The cell wall's strength is enhanced by teichoic acids, glycopolymeric substances embedded within the peptidoglycan. Teichoic acids play multiple roles, such as generating the cell's net negative charge, contributing to cell wall rigidity and shape maintenance, and aiding in cell division and resistance to various stressors, including heat and salt. Despite the density of the peptidoglycan layer, it remains relatively porous, allowing most substances to permeate. For larger nutrients, Gram-positive bacteria utilize exoenzymes, secreted extracellularly to break down macromolecules outside the cell.{{Cite book |last=Bruslind |first=Linda |url=https://open.oregonstate.education/generalmicrobiology/ |title=General Microbiology |date=2019-08-01 |publisher=Oregon State University |language=en}}

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 Lactobacillus, Bacillus, Listeria, Staphylococcus, Streptococcus, Enterococcus, and Clostridium.{{Cite journal |last=Galperin |first=Michael Y. |date=27 December 2013 |title=Genome Diversity of Spore-forming Firmicutes |journal=Microbiology Spectrum |volume=1 |issue=2 |pages=TBS-0015-2012- |doi=10.1128/microbiolspectrum.tbs-0015-2012 |issn=2165-0497 |pmc=4306282 |pmid=26184964}} It has also been expanded to include the Mollicutes, bacteria such as Mycoplasma and Thermoplasma that lack cell walls and so cannot be Gram-stained, but are derived from such forms.{{cite web |last=Hashem |first=Hams H. |title=Practical Medical Microbiology |work=University of Al-Qadisiya |url=http://qu.edu.iq/el/mod/resource/view.php?id=1391 }}{{Dead link|date=June 2024 |bot=InternetArchiveBot |fix-attempted=yes }}

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 procedures.{{Cite web |url= http://www2.highlands.edu/academics/divisions/scipe/biology/labs/rome/acid_fast_stain.htm |title=The Acid Fast Stain |work=www2.Highlands.edu |publisher=Georgia Highlands College |access-date=10 June 2017 |archive-url= https://web.archive.org/web/20170610135044/http://www2.highlands.edu/academics/divisions/scipe/biology/labs/rome/acid_fast_stain.htm |archive-date=10 June 2017 |url-status=dead}}

{{Main|Gram-negative bacteria}}

File:Neisseria gonorrhoeae and pus cells Gram stain.jpg and pus cells]]

Gram-negative bacteria generally possess a thin layer of peptidoglycan between two membranes (diderm).{{cite journal | vauthors=Megrian D, Taib N, Witwinowski J, Gribaldo S| title=One or two membranes? Diderm Firmicutes challenge the Gram-positive/Gram-negative divide | journal= Molecular Microbiology | volume=113 | issue=3 | pages=659–671 | year=2020 | url=https://hal.archives-ouvertes.fr/pasteur-02505848 | doi= 10.1111/mmi.14469 | pmid=31975449 | s2cid=210882600 | doi-access=free }} Lipopolysaccharide (LPS) is the most abundant antigen on the cell surface of most gram-negative bacteria, contributing up to 80% of the outer membrane of E. coli and Salmonella.{{cite journal | vauthors=Avila-Calderón ED, Ruiz-Palma MD, Contreras-Rodríguez A| title=Outer Membrane Vesicles of Gram-Negative Bacteria: An Outlook on Biogenesis | journal= Frontiers in Microbiology | volume=12 | pages=557902 | year=2021 | doi= 10.3389/fmicb.2021.557902 | pmc=7969528 | pmid=33746909 | doi-access=free }} These LPS molecules, consisting of the O-antigen or O-polysaccharide, core polysaccharide, and lipid A, serve multiple functions including contributing to the cell's negative charge and protecting against certain chemicals. LPS's role is critical in host-pathogen interactions, with the O-antigen eliciting an immune response and lipid A acting as an endotoxin.

Additionally, the outer membrane acts as a selective barrier, regulated by porins, transmembrane proteins forming pores that allow specific molecules to pass. The space between the cell membrane and the outer membrane, known as the periplasm, contains periplasmic enzymes for nutrient processing. A significant structural component linking the peptidoglycan layer and the outer membrane is Braun's lipoprotein, which provides additional stability and strength to the bacterial cell wall.

Most bacterial phyla are gram-negative, including the cyanobacteria, green sulfur bacteria, and most Pseudomonadota (exceptions being some members of the Rickettsiales and the insect-endosymbionts of the Enterobacteriales).{{page needed|date=March 2016}}

{{anchor|Gram-variable and gram-indeterminate bacteria|Gram-indeterminate bacteria|reason=Former names of this section; may have incoming links.}}

Some bacteria, after staining with the Gram stain, yield a gram-variable pattern: a mix of pink and purple cells are seen.{{cite journal |url= |first=Terry J. |last=Beveridge |title=Mechanism of Gram Variability in Select Bacteria |journal=Journal of Bacteriology |date=March 1990 |volume=172 |issue=3 |pages=1609–1620 |pmid=1689718 |pmc=208639 |doi=10.1128/jb.172.3.1609-1620.1990}} 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 stain.

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 bovis, Mycobacterium leprae and Mycobacterium tuberculosis, the latter two of which are the causative agents of leprosy and tuberculosis, respectively.{{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}}{{cite book |last1=Reynolds |first1=J. |last2=Moyes |first2=R. B. |last3=Breakwell |first3=D. P. |title=Differential staining of bacteria: Acid fast stain |journal=Current Protocols in Microbiology |date=2009 |chapter=Appendix 3 |volume=Appendix 3 |pages=H |doi=10.1002/9780471729259.mca03hs15 |pmid=19885935 |isbn=978-0471729259|s2cid=45685776 }} Bacteria of the genus Mycoplasma lack a cell wall around their cell membranes,{{cite book | author = Ryan KJ, Ray CG (editors) | title = Sherris Medical Microbiology | pages=409–12 |edition = 4th | publisher = McGraw Hill | year = 2004 | isbn = 978-0-8385-8529-0}} which means they do not stain by Gram's method and are resistant to the antibiotics that target cell wall synthesis.{{cite journal |vauthors=Lee EH, Winter HL, van Dijl JM, Metzemaekers JD, Arends JP |title=Diagnosis and antimicrobial therapy of Mycoplasma hominis meningitis in adults |journal=International Journal of Medical Microbiology |volume=302 |issue=7–8 |pages=289–92 |date=December 2012 |pmid=23085510 |doi=10.1016/j.ijmm.2012.09.003 |url=}}{{cite journal |vauthors=Gautier-Bouchardon AV |title=Antimicrobial Resistance in Mycoplasma spp |journal=Microbiology Spectrum |volume=6 |issue=4 |pages= 425–446|date=July 2018 |pmid=30003864 |doi=10.1128/microbiolspec.ARBA-0030-2018 |isbn=9781555819798 |s2cid=51616821 |url=|pmc=11633602 }}

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.{{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=Oxford University Press |date=28 August 2014 |isbn=978-0199570027 }} The initial letters of gram-positive and gram-negative, which are eponymous adjectives, can be either capital G or lowercase g, depending on what 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.{{cite web |url= http://wwwnc.cdc.gov/eid/pages/preferred-usage.htm |title=Preferred Usage |work=Emerging Infectious Diseases Style Guide |publisher=Centers for Disease Control and Prevention}} Dictionaries may use lowercase,{{cite web |title=Dorland's Illustrated Medical Dictionary |edition=32nd |publisher=Elsevier |url= https://www.dorlands.com/dorlands/wsearch.jsp |access-date=5 June 2020}} Use search terms such as {{kbd|gram-negative}}.{{Citation |title=gram–positive |work=Merriam-Webster |publisher=Encyclopædia Britannica, Inc. |url= http://www.merriam-webster.com/dictionary/gram%E2%80%93positive}} uppercase,{{cite web |url= http://www.collinsdictionary.com/dictionary/english/gram-positive |title=Gram-positive |work=CollinsDictionary.com |publisher=HarperCollins}}{{cite web |url= https://www.lexico.com/definition/gram_stain |archive-url= https://web.archive.org/web/20200605094227/https://www.lexico.com/definition/gram_stain |url-status= dead |archive-date= June 5, 2020 |title=Gram stain |work=Lexico.com |publisher=Oxford University Press}}{{cite web |url= http://www.medicinenet.com/script/main/art.asp?articlekey=9585 |title=Gram-positive |work=MedicineNet}}{{cite web |url= http://www.businessdictionary.com/definition/Gram-negative-positive.html |title= Gram negative/positive |work= BusinessDictionary.com |access-date= 2016-10-20 |archive-date= 2016-10-20 |archive-url= https://web.archive.org/web/20161020173301/http://www.businessdictionary.com/definition/Gram-negative-positive.html |url-status= dead }} or both.{{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 |publisher=Houghton Mifflin}}{{cite web |url= http://www.dictionary.com/browse/gram-positive |title=Gram-positive |work=Dictionary.com}} Uppercase Gram-positive or Gram-negative usage is also common in many scientific journal articles and publications.{{cite journal |title=Through the wall: Extracellular vesicles in Gram-positive bacteria, mycobacteria and fungi |first1=Lisa |last1=Brown |first2=Julie M. |last2=Wolf |first3=Rafael |last3=Prados-Rosales |first4=Arturo |last4=Casadevall |journal=Nature Reviews Microbiology |volume=13 |issue=10 |pages=620–630 |date=2015 |doi=10.1038/nrmicro3480 |pmid=26324094 |pmc=4860279}}{{cite journal |journal=Science |title=Detecting Gram-negative bacteria |date=12 June 2015 |volume=348 |issue=6240 |page=1218 |doi=10.1126/science.348.6240.1218-o |first=Kristen L. |last=Mueller}} 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 typically use capital for Gram stain.{{cn|date=May 2024}}

• Bacterial cell structure
• Ziehl–Neelsen stain

{{Reflist}}

{{Commons category|Gram stains}}

{{Wikibooks|School Science|Gram staining}}

• [http://www.tgw1916.net/movies.html Gram staining technique video]

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