Editing Staining (section)

== Preparation ==
The preparatory steps involved depend on the type of analysis planned. Some or all of the following procedures may be required.

'''[[Wet mount]]s''' are used to view live organisms and can be made using water and certain stains. The liquid is added to the slide before the addition of the organism and a coverslip is placed over the specimen in the water and stain to help contain it within the [[field of view]].<ref name=":1">{{cite book |last=Parker |first=Nina | name-list-style = vanc |title=Microbiology|publisher=OpenStax|year=2012}}</ref>

'''[[Fixation (histology)|Fixation]]''', which may itself consist of several steps, aims to preserve the shape of the cells or tissue involved as much as possible.  Sometimes [[heat fixation]] is used to kill, adhere, and alter the specimen so it accepts stains. Most chemical fixatives (chemicals causing fixation) generate [[chemical bond]]s between [[protein]]s and other substances within the sample, increasing their rigidity.  Common fixatives  include [[formaldehyde]], [[ethanol]], [[methanol]], and/or [[picric acid]].  Pieces of tissue may be embedded in [[paraffin wax]] to increase their mechanical strength and stability and to make them easier to cut into thin slices.<ref name=":0">{{cite book | vauthors = Pommerville JC |title= Fundamentals of Microbiology | volume = I |publisher= Jones & Bartlett Learning |year= 2017 |pages=248, 249|isbn = 978-1-284-10095-2 }}</ref>

'''[[Mordant]]s''' are chemical agents which have power of making dyes to stain materials which otherwise are unstainable

Mordants are classified into two categories:

a) Basic mordant: React with acidic dyes e.g. alum, ferrous sulfate, cetylpyridinium chloride etc.

b) Acidic mordant : React with basic dyes e.g. picric acid, tannic acid etc.

<ref name=":0" />'''Direct Staining:''' Carried out without mordant.

'''Indirect Staining:''' Staining with the aid of a mordant.
{| class="wikitable"
|+Table represents Indirect Staining Techniques and mordants applied in each:
!Sr No.
!Name of Indirect Staining Technique
!Name of mordant applied
|-
|1.)
|Gram's Staining
|Gram's iodine
|-
|2.)
|Cell Wall Staining
a.) Ringer's method

b.) Dyar's method
|10% Tannic acid

0.34% C.P.C
|-
|3.)
|Flagella Staining
a.) Leifson's method

b.) Loeffler's method
|Tannic acid in Leifson's stain

Loeffler's mordant (20%Tannic acid )
|-
|4.)
|Spirochete Staining
a.) Fontana's method

b.) Becker's method
|Fontana's mordant(5%Tannic acid)

Fontana's mordant(5%Tannic acid)
|}
'''Permeabilization''' involves treatment of cells with (usually) a mild [[surfactant]].  This treatment dissolves [[cell membrane]]s, and allows larger dye molecules into the cell's interior.

'''[[Microscope slide#Mounting|Mounting]]''' usually involves attaching the samples to a glass microscope slide for observation and analysis.  In some cases, cells may be grown directly on a slide.  For samples of loose cells (as with a blood smear or a [[pap smear]]) the sample can be directly applied to a slide.  For larger pieces of tissue, thin sections (slices) are made using a [[microtome]]; these slices can then be mounted and inspected.

=== Standardization ===

Most of the dyes commonly used in microscopy are available as '''BSC-certified stains'''. This means that samples of the manufacturer's batch have been tested by an independent body, the [[Biological Stain Commission]] ('''BSC'''), and found to meet or exceed certain standards of purity, dye content and performance in staining techniques ensuring more accurately performed experiments and more reliable results. These standards are published in the commission's journal [[Biotechnic & Histochemistry]].<ref>{{cite journal | vauthors = Penney DP, Powers JM, Frank M, Willis C, Churukian C | title = Analysis and testing of biological stains--the Biological Stain Commission Procedures | journal = Biotechnic & Histochemistry | volume = 77 | issue = 5–6 | pages = 237–75 | year = 2002 | pmid = 12564600 | doi = 10.1080/714028210 }}</ref> Many dyes are inconsistent in composition from one supplier to another. The use of BSC-certified stains eliminates a source of unexpected results.<ref name="HorobinKiernan" />

Some vendors sell stains "certified" by themselves rather than by the Biological Stain Commission. Such products may or may not be suitable for diagnostic and other applications.<ref>{{cite web|url=https://biologicalstaincommission.org/vendors-list/|title=Vendors List - The Biological Stain Commission|website=biologicalstaincommission.org|access-date=25 March 2018}}</ref>

=== Negative staining ===
[[File:HexLamMic phases.jpg|thumb|Example of [[negative staining]]]]
{{main|Negative staining}}
A simple staining method for bacteria that is usually successful, even when the [[#Positive staining|positive staining]] methods fail, is to use a [[negative stain]]. This can be achieved by smearing the sample onto the slide and then applying [[nigrosin]] (a black synthetic dye) or [[India ink]] (an aqueous suspension of carbon particles). After drying, the microorganisms may be viewed in bright field microscopy as lighter inclusions well-contrasted against the dark environment surrounding them.<ref>{{cite book | vauthors = Clark G | date = 1981 | title = Staining Procedures | edition = 4th | location = Baltimore | publisher = Williams & Wilkins | page = 412 | isbn = 978-0-683-01707-6 }}</ref>  Negative staining is able to stain the background instead of the organisms because the cell wall of microorganisms typically has a negative charge which repels the negatively charged stain. The dyes used in negative staining are acidic.<ref name=":1" /> Note: negative staining is a mild technique that may not destroy the microorganisms, and is therefore unsuitable for studying pathogens.

=== Positive staining ===
Unlike negative staining, positive staining uses basic dyes to color the specimen against a bright background. While [[chromophore]] is used for both negative and positive staining alike, the type of chromophore used in this technique is a positively charged ion instead of a negative one. The negatively charged cell wall of many microorganisms attracts the positively charged chromophore which causes the specimen to absorb the stain giving it the color of the stain being used. Positive staining is more commonly used than negative staining in microbiology. The different types of positive staining are listed below.<ref name=":1" />

=== Simple versus differential ===
Simple Staining is a technique that only uses one type of stain on a slide at a time. Because only one stain is being used, the specimens (for positive stains) or background (for negative stains) will be one color. Therefore, simple stains are typically used for viewing only one organism per slide. Differential staining uses multiple stains per slide. Based on the stains being used, organisms with different properties will appear different colors allowing for categorization of multiple specimens. Differential staining can also be used to color different organelles within one organism which can be seen in [[endospore staining]].<ref name=":1" />

=== Types ===
{| class="wikitable"
|+ Types of staining techniques<ref>{{Cite book|title=Elementary Microbiology Vol - I}}</ref>
!Sr. No.
!Staining Technique
!Preparation
!Application
!Result
|-
|1.
|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
|-
|2.
|Negative (Relief)
|Smear mixed with Nigrosin and spread
into thin film
|Study cell morphology
|Organism is stained, the background is black
|-
|3
|Gram
|Primary stain: Crystal violet applied to film then treated with iodine (mordant), alcohol (decolourizer) and counter stained with safranin
|Characterizes bacteria in one of two groups, Gram positive or Gram negative
|Gram positive appears purple in color
Gram negative appears pink in color
|-
|4
|Acid fast (Ziehl-Neelsen technique)
|Film stained with hot Z.N.C.F. decolourised (acid-alcohol) and counter stain with methylene blue
|Separate non-decolorized acid fast bacteria that are not decolorized from colorized non-acid fast bacteria
|Acid fast bacteria:Red
Non acid fast: Blue
|-
|5
|Endospore (Dornor's method)
|Primary stain Malachite green heat fixed to penetrate spores; vegetative cells are counterstained with Safranin
|Detects the presence of endospores in six genera of bacteria
|Endospores: Green
Vegetative cells: Red
|-
|6
|Capsule
A: Hiss method (Positive technique)

B: Manevals's technique (Negative)
|Smear stained with Hiss stain following treatment with copper sulphate

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/pale mauve color
Bacteria: Purple capsule, bacterial cell, stands out against dark background
|-
|7
|Cell wall (Dyar's method)
|Smear treated with C.P.C. which dissociates to form positively charged cetyl pyridinium and negatively charged chloride ions. Positively charged ions are adsorbed on negatively charged cell wall
|Stains cell wall of bacterium
|Cell wall: Red Cytoplasm: Blue
|-
|8
|Flagella (Leifson's method)
|Mordant acts to thicken flagella before staining and increases visibility microscopically when stained with Leifson stain
|Demonstrates presence of flagella
|Flagella: Red Vegetative cells: Blue
|-
|9
|Nuclear material (Feulgen technique)
|Smear is treated for hydrolysis to release purines from DNA, purines to cause shift form furanose to aldehyde. Aldehyde groups are available to react with schiff's reagent to form addition compounds.
|To demonstrate the presence of DNA in cell. But for detection of the DNA, RNA should be selectively destroyed by acid hydrolysis without affecting DNA
|Nuclear material- pinkish purple,
Cytoplasm- colorless
|-
|10
|Metachromatic granules (Alberts's method)
|The smear is first treated with chloroform to remove fats . Smear applied with Alberts stain which contains cationic dyes such as toluidine blue and malachite green. Toluidine blue preferentially stains granules while malachite green stains cytoplasm.
|The granules show the typical monochromatism nature, this is used to demonstrate granules
|Granules: Bluish black, Cytoplasm: Green
|-
|11
|Intracellular lipids (Burdon's method)
|Lipids are stained with fat soluble dyes like Sudan black. On application of Sudan black-B dyes move into lipids and are retained there while cytoplasm is counter stained with safranin.
|To detect the presence of lipids in cell wall, cell membrane or fat globules (PHB in cytoplasm)
|Lipid granules: Deep blue,
Cytoplasm: Light pink
|-
|12
|Polysaccharide (Hotch kuss method)
|Polysaccharide is oxidized with periodate to form polyaldehyde which reacts with Schiff's reagents to red color, while cytoplasm is counter stained with malachite green
|Detects the accumulation of polysaccharide granules in the cells
|Polysaccharide: Red
Cytoplasm: Green
|}