Editing Staining (section)

== Techniques ==

=== Gram ===
{{main|Gram staining}}
[[Gram staining]] is used to determine gram status to classifying bacteria broadly based on the composition of their [[cell wall]]. Gram staining uses [[Gentian violet|crystal violet]] to stain cell walls, [[iodine]] (as a mordant), and a [[fuchsin]] or [[safranin]] counterstain to (mark all bacteria). Gram status, helps divide specimens of bacteria into two groups, generally representative of their underlying phylogeny. This characteristic, in combination with other techniques makes it a useful tool in clinical microbiology laboratories, where it can be important in early selection of appropriate [[antibiotic]]s.<ref>{{Cite journal|last1=Stone|first1=Rebecca B.|last2=Steele|first2=John C. H.|date=2009-07-01|title=Impact of Reporting Gram Stain Results From Blood Cultures on the Selection of Antimicrobial Agents|journal=American Journal of Clinical Pathology|language=en|volume=132|issue=1|pages=5–6|doi=10.1309/AJCP9RUV0YGLBVHA|pmid=19864226|issn=0002-9173|doi-access=free}}</ref>

<!-- Since this is a summary article, the following paragraph is probably more appropriate for the main article. Also, this explanation is one of two main theories. I think neither has been empirically demonstrated to date (see https://academic.oup.com/ajcp/article/132/1/5/1765499). -->On most Gram-stained preparations, [[Gram-negative]] organisms appear red or pink due to their counterstain. Due to the presence of higher lipid content, after alcohol-treatment, the porosity of the cell wall increases, hence the CVI complex (crystal violet – iodine) can pass through. Thus, the primary stain is not retained. In addition, in contrast to most Gram-positive bacteria, Gram-negative bacteria have only a few layers of peptidoglycan and a secondary cell membrane made primarily of lipopolysaccharide.

=== Endospore ===
{{main|Endospore staining}}
[[Endospore staining]] is used to identify the presence or absence of [[endospore]]s, which make bacteria very difficult to kill.  Bacterial spores have proven to be difficult to stain as they are not permeable to aqueous dye reagents.&nbsp; Endospore staining is particularly useful for identifying endospore-forming bacterial [[pathogen]]s such as ''[[Clostridioides difficile (bacteria)|Clostridioides difficile]]''. Prior to the development of more efficient methods, this stain was performed using the Wirtz method with heat fixation and counterstain. Through the use of malachite green and a diluted ratio of carbol fuchsin, fixing bacteria in osmic acid was a great way to ensure no blending of dyes. However, newly revised staining methods have significantly decreased the time it takes to create these stains. This revision included substitution of carbol fuchsin with aqueous Safranin paired with a newly diluted 5% formula of malachite green. This new and improved composition of stains was performed in the same way as before with the use of heat fixation, rinsing, and blotting dry for later examination. Upon examination, all endospore forming bacteria will be stained green accompanied by all other cells appearing red.<ref>{{cite journal | vauthors = Schaeffer AB, Fulton MD | title = A Simplified Method of Staining Endospores | journal = Science | volume = 77 | issue = 1990 | pages = 194 | date = February 1933 | pmid = 17741261 | doi = 10.1126/science.77.1990.194 | bibcode = 1933Sci....77..194S }}</ref>

=== Ziehl-Neelsen ===
{{main|Ziehl–Neelsen stain}}

A [[Ziehl–Neelsen stain]] is an acid-fast stain used to stain species of ''[[Mycobacterium tuberculosis]]'' that do not stain with the standard laboratory staining procedures such as Gram staining.

This stain is performed through the use of both red coloured [[carbol fuchsin]] that stains the bacteria and a counter stain such as [[methylene blue]].

=== Haematoxylin and eosin (H&E) ===
{{main|H&E stain}}
[[Image:Emphysema H and E.jpg|right|thumb|Microscopic view of a histologic specimen of human [[lung]] tissue stained with [[hematoxylin]] and [[eosin]].]]
[[H&E stain|Haematoxylin and eosin stain]]ing is frequently used in [[histology]] to examine thin tissue sections.<ref name="Bancroft and Stevens, 1982">{{cite book|title=The Theory and Practice of Histological Techniques|date=1982|publisher=Longman Group Limited |editor1-last=Bancroft |editor2-last=Stevens|editor2-first=Alan | name-list-style = vanc |editor1-first=John|edition=2nd}}</ref>  [[Haematoxylin]] stains cell nuclei blue, while [[eosin]] stains cytoplasm, connective tissue and other extracellular substances pink or red.<ref name="Bancroft and Stevens, 1982" />  Eosin is strongly absorbed by [[red blood cell]]s, colouring them bright red. In a skillfully made H&E preparation the red blood cells are almost orange, and collagen and cytoplasm (especially muscle) acquire different shades of pink.

=== 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 ===
{{main|Periodic acid–Schiff stain}}
[[Image:Histoplasma pas-d small.jpg|thumb|right|[[PAS diastase]] showing the fungus [[Histoplasma]].]]
<ref>{{Cite web|url=https://labce.com/spg949466_periodic_acid_schiff_pas_diagnostic_applications.aspx|title=Periodic Acid-Schiff (PAS): Diagnostic Applications - LabCE.com, Laboratory Continuing Education|website=labce.com|access-date=2020-04-16}}</ref>[[Periodic acid-Schiff]] is a histology special stain used to mark [[carbohydrate]]s ([[glycogen]], [[glycoprotein]], [[proteoglycan]]s). PAS is commonly used on liver tissue where glycogen deposits are made which is done in efforts to distinguish different types of glycogen storage diseases. PAS is important because it can detect glycogen granules found in tumors of the ovaries and pancreas of the endocrine system, as well as in the bladder and kidneys of the renal system. Basement membranes can also show up in a PAS stain and can be important when diagnosing renal disease. Due to the high volume of carbohydrates within the cell wall of hyphae and yeast forms of fungi, the Periodic acid -Schiff stain can help locate these species inside tissue samples of the human body.

=== Masson ===
{{main|Masson's trichrome stain}}
[[Masson's trichrome]] is (as the name implies) a three-colour staining protocol.  The recipe has evolved from Masson's original technique for different specific applications, but all are well-suited to distinguish cells from surrounding [[connective tissue]].  Most recipes produce red [[keratin]] and muscle fibers, blue or green staining of [[collagen]] and [[bone]], light red or pink staining of [[cytoplasm]], and black [[cell nucleus|cell nuclei]].

=== Romanowsky ===
{{main|Romanowsky stain}}
The [[Romanowsky stain]]s is considered a polychrome staining effect and is based on a combination of eosin plus (chemically [[reduction (chemistry)|reduced]] [[eosin]]) and demethylated [[methylene blue]] (containing its oxidation products [[azure A]] and [[azure B]]).  This stain develops varying colors for all cell structures (“Romanowsky-Giemsa effect) and thus was used in staining neutrophil polymorphs and cell nuclei. Common variants include [[Wright's stain]], [[Jenner's stain]], May-Grunwald stain, [[Leishman stain]] and [[Giemsa stain]].

All are used to examine [[blood]] or [[bone marrow]] samples.  They are preferred over H&E for inspection of blood cells because different types of [[white blood cells|leukocytes]] (white blood cells) can be readily distinguished.  All are also suited to examination of blood to detect blood-borne parasites such as [[malaria]].<ref name = "Bezrukov_2017">{{cite journal | vauthors = Bezrukov AV | title = Romanowsky staining, the Romanowsky effect and thoughts on the question of scientific priority | journal = Biotechnic & Histochemistry | volume = 92 | issue = 1 | pages = 29–35 | date = 2017-01-02 | pmid = 28098484 | doi = 10.1080/10520295.2016.1250285 | s2cid = 37401579 }}</ref>

=== Silver ===
[[Image:Histoplasma in granuloma gms.jpg|thumb|right|[[Gömöri methenamine silver stain]] demonstrating [[histoplasma]] (illustrated in black).]]
[[Silver stain]]ing is the use of [[silver]] to stain [[histologic section]]s. This kind of staining is important in the demonstration of [[protein]]s (for example type III [[collagen]]) and [[DNA]]. It is used to show both substances inside and outside [[Cell (biology)|cells]]. Silver staining is also used in [[temperature gradient gel electrophoresis]].

''Argentaffin cells'' [[Redox|reduce]] silver solution to metallic silver after [[formalin]] [[fixation (histology)|fixation]]. This method was discovered by Italian [[Camillo Golgi]], by using a reaction between [[silver nitrate]] and [[potassium dichromate]], thus precipitating silver chromate in some cells (see [[Golgi's method]]). A''rgyrophilic cells'' reduce silver solution to metallic silver after being exposed to the stain that contains a [[Reducing agent|reductant]]. An example of this would be [[hydroquinone]] or formalin.

=== Sudan ===
{{main|Sudan stain}}
[[Sudan stain]]ing utilizes Sudan dyes to stain sudanophilic substances, often including [[lipid]]s. [[Sudan III]], [[Sudan IV]], [[Oil Red O]], [[Osmium tetroxide]], and [[Sudan Black B]] are often used. Sudan staining is often used to determine the level of [[fecal fat]] in diagnosing [[steatorrhea]].

=== Wirtz-Conklin ===
The Wirtz-Conklin stain is a special technique designed for staining true endospores with the use of malachite green dye as the primary stain and safranin as the counterstain. Once stained, they do not decolourize. The addition of heat during the staining process is a huge contributing factor.<ref>{{cite journal | vauthors = Corey L | title = Laboratory diagnosis of herpes simplex virus infections. Principles guiding the development of rapid diagnostic tests | journal = Diagnostic Microbiology and Infectious Disease | volume = 4 | issue = 3 Suppl | pages = 111S–119S | date = March 1986 | pmid = 3009082 | doi = 10.1016/s0732-8893(86)80049-9 }}</ref> Heat helps open the spore's membrane so the dye can enter. The main purpose of this stain is to show germination of bacterial spores. If the process of germination is taking place, then the spore will turn green in color due to malachite green and the surrounding cell will be red from the safranin. This stain can also help determine the orientation of the spore within the bacterial cell; whether it being terminal (at the tip), subterminal (within the cell), or central (completely in the middle of the cell).

===Collagen hybridizing peptide===
{{main|Collagen hybridizing peptide}}
[[Collagen hybridizing peptide]] (CHP) staining allows for an easy, direct way to stain denatured collagens of any type (Type I, II, IV, etc.) regardless if they were damaged or degraded via enzymatic, mechanical, chemical, or thermal means. They work by refolding into the collagen triple helix with the available single strands in the tissue. CHPs can be visualized by a simple [[fluorescence microscope]].