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''[[In vitro]]'' staining involves colouring cells or structures that have been removed from their biological context. Certain stains are often combined to reveal more details and features than a single stain alone. Combined with specific protocols for [[fixation (histology)|fixation]] and sample preparation, scientists and physicians can use these standard techniques as consistent, repeatable diagnostic tools. A [[counterstain]] is stain that makes cells or structures more visible, when not completely visible with the principal stain.
* Crystal violet stains both Gram positive and Gram negative organisms. Treatment with alcohol removes the crystal violet colour from gram negative organisms only. [[Safranin]] as counterstain is used to colour the gram negative organisms that got decolorised by alcohol.
While ex vivo, many cells continue to live and metabolize until they are "fixed". Some staining methods are based on this property. Those stains excluded by the living cells but taken up by the already dead cells are called [[vital stain]]s (e.g. [[trypan blue]] or [[propidium iodide]] for eukaryotic cells). Those that enter and stain living cells are called [[supravital stain]]s (e.g. [[New Methylene Blue]] and [[brilliant cresyl blue]] for [[reticulocyte]] staining). However, these stains are eventually toxic to the organism, some more so than others. Partly due to their toxic interaction inside a living cell, when supravital stains enter a living cell, they might produce a characteristic pattern of staining different from the staining of an already fixed cell (e.g. "reticulocyte" look versus diffuse "polychromasia"). To achieve desired effects, the stains are used in very dilute solutions ranging from {{gaps|1|:|5|000}} to {{gaps|1|:|500|000}} (Howey, 2000). Note that many stains may be used in both living and fixed cells.
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|Simple (Monochrome)
|Smear stain with single dye .
e.g. Methylene blue, Safranin°≤×←→ etc.
|Used to highlight microbes and illustrate cellular
shapes and arrangements
|Organisms are stained in the color of applied stain
|-
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|Characterizes bacteria in one of two groups, Gram positive or Gram negative
|Gram positive appears purple in color
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|Acid fast (Ziehl-Neelsen technique)
|Film stained with hot Z.N.C.F.
|Separate non-decolorized acid fast bacteria that are not decolorized from colorized non-acid fast bacteria
|Acid fast bacteria:Red
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Bacterial suspension smeared along with Congo red and the Maneval's stain is applied
|Capsules can be observed as clear zones surrounding cells of capsulated bacteria and are used to demonstrate the presence of capsules.
|Capsule: Light violet/
Bacteria: Purple capsule, bacterial cell,
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=== Papanicolaou ===
{{main|Papanicolaou stain}}
[[Papanicolaou stain]]ing, or PAP staining, was developed to replace fine needle aspiration cytology (FNAC) in hopes of decreasing staining times and cost without compromising quality. This stain is a frequently used method for examining cell samples from a variety of tissue types in various organs. PAP staining has endured several modifications in order to become a “suitable alternative” for FNAC. This transition stemmed from the appreciation of wet fixed smears by scientists preserving the structures of the nuclei opposed to the opaque appearance of air dried Romanowsky smears. This led to the creation of a hybrid stain of wet fixed and air dried known as the ultrafast papanicolaou stain. This modification includes the use of nasal saline to rehydrate cells to increase cell transparency and is paired with the use of alcoholic formalin to enhance colors of the nuclei. The papanicolaou stain is now used in place of cytological staining in all organ types due to its increase in morphological quality, decreased staining time, and decreased cost. It is frequently used to stain [[Pap smear]] specimens.<ref name="Gill, 2013">{{cite book|last1=Gill|first1=Gary W. | name-list-style = vanc |title=Cytopreparation|year=2013|isbn=978-1-4614-4932-4|series=Essentials in Cytopathology|volume=12|pages=143–189|chapter=Papanicolaou Stain|doi=10.1007/978-1-4614-4933-1_10|issn=1574-9053}}</ref> It uses a combination of [[haematoxylin]], [[Orange G]], [[eosin Y]], [[Light Green SF yellowish]], and sometimes [[Bismarck Brown Y]].<ref name="Bancroft and Stevens, 1982" /><ref name="Gill, 2013" /><ref>{{cite journal | vauthors = Thakur M, Guttikonda VR | title = Modified ultrafast Papanicolaou staining technique: A comparative study | journal = Journal of Cytology | volume = 34 | issue = 3 | pages = 149–153 | date = 2017 | pmid = 28701828 | pmc = 5492752 | doi = 10.4103/JOC.JOC_23_16 | doi-access = free }}</ref>
=== PAS ===
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===Collagen hybridizing peptide===
{{main|Collagen hybridizing peptide}}
[[Collagen
== Common biological stains ==
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=== Bismarck brown ===
<ref>{{Cite journal| vauthors = Tomov N, Dimitrov N |date=2017|title=Modified bismarck brown staining for demonstration of soft tissue mast cells |url= https://tru.uni-sz.bg/tsj/Vol15_N3_2017/2_N.Tomov.pdf|journal=Trakia Journal of Sciences |volume=15 |issue=3 |pages=195–197 |doi=10.15547/tjs.2017.03.001 |doi-access=free }}</ref>[[Bismarck brown Y|Bismarck brown]] (also Bismarck brown Y or Manchester brown) imparts a yellow colour to acid [[mucin]]s
=== Carmine ===
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=== Coomassie blue ===
[[Coomassie brilliant
===Cresyl violet===
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=== Methyl green ===
[[Methyl green]] is used commonly with bright-field, as well as fluorescence microscopes <ref>{{cite journal | vauthors = Prieto D, Aparicio G, Morande PE, Zolessi FR | title = A fast, low cost, and highly efficient fluorescent DNA labeling method using methyl green | journal = Histochemistry and Cell Biology | volume = 142 | issue = 3 | pages = 335–45 | date = September 2014 | pmid = 24671497 | doi = 10.1007/s00418-014-1215-0 | hdl = 11336/35891 | s2cid = 11094194 | hdl-access = free }}</ref> to dye the chromatin of cells so that they are more easily viewed.
=== Methylene blue ===
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=== Safranine ===
[[Safranine]] (or Safranine O) is a red cationic dye. It binds to nuclei (DNA) and other tissue [[polyanions]], including
The incorrect spelling "safranin" is in common use. The -ine ending is appropriate for safranine O because this dye is an amine.<ref name="HorobinKiernan">{{cite book |isbn=978-1-85996-099-8 |title=Conn's Biological Stains: A Handbook of Dyes, Stains and Fluorochromes for Use in Biology and Medicine |year=2002 |editor-last1 = Horobin | editor-first1 = Richard | editor-last2 = Kiernan | editor-first2 = John | name-list-style = vanc |publisher=Taylor & Francis }}</ref><ref>Baker JR (1958). ''Principles of Biological Microtechnique''. pp. 329 ff. London: Methuen.</ref><ref>{{cite journal | vauthors = Kiernan JA | title = Classification and naming of dyes, stains and fluorochromes | journal = Biotechnic & Histochemistry | volume = 76 | issue = 5–6 | pages = 261–78 | year = 2001 | pmid = 11871748 | doi = 10.1080/bih.76.5-6.261.278 | s2cid = 32479873 }}</ref>
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* {{cite book | veditors = Bancroft JD, Gamble M | date = 2002 | title = Theory and Practice of Histological Techniques | edition = 5th | location = London | publisher = Churchill-Livingstone | isbn = 978-0-443-06435-7 }}
* {{cite book | vauthors = Kiernan JA | date = 2015 | title = Histological and Histochemical Methods. Theory and Practice | location = Banbury, UK | publisher = Scion | isbn = 978-1-907904-32-5 }}
* {{cite book | vauthors = Presnell JK, Schreibman MP | date = 1997 | title = Humason's Animal tissue Techniques | url = https://archive.org/details/humasonsanimalti0000pres | url-access = registration | edition = 5th | location = Baltimore | publisher = Johns Hopkins University Press | isbn = 9780801854019 }}
* {{cite book | vauthors = Ruzin SE | date = 1999 | title = Plant Microtechnique and Microscopy | location = New York | publisher = Oxford University Press | isbn = 978-0-19-508956-1 }}
{{refend}}
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* [https://www.stainsfile.com/ StainsFile] Reference for dyes and staining techniques.
* [https://www.microscopy-uk.org.uk/mag/artfeb00/rhvital.html Vital Staining for Protozoa and Related Temporary Mounting Techniques] ~ Howey, 2000
* [
* [https://www.histology-world.com/stains/stains.htm Photomicrographs of Histology Stains]
* [https://www.microrao.com/staining.htm Frequently asked questions in staining exercises] at Sridhar Rao P.N's home page
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