Staining Technique

• Staining is a method used to boost the contrast of samples, usually at the microscopic scale.
• Colors and staining are often utilized in the field of histology (microscopic examination of living tissues) as well as in areas of medical histopathology, Hematology, and Cytopathology which are focused on studying and diagnosis of disease at a microscopic scale.
• Stains are used to identify biological tissues (highlighting the, for instance muscles fibers or connective tissue) Cell populations (classifying various blood cells) or organelles within each cell.
• In biochemistry, it is the process of the addition of a specific class (DNA proteins, lipids or carbohydrates) dye to a substrate in order to determine or measure whether or not it is a particular chemical.
• Biological staining is also employed to distinguish cells during flow cytometry and also to identify the presence of nucleic acids or proteins in electrophoresis gel.
• Light microscopes are utilized to examine stained samples with high magnification. Typically, they use epi-fluorescence or bright field illumination.
• Staining does not have to be restricted to biological materials. It can be utilized to analyze the structure of other materials , for example , the lamellar structures of semi-crystalline polymers, or the structures of the domains of block copolymers.

Types of Staining Techniques

A. Simple staining (Monochrome)

• In this method the smear of bacteria is stained using a single reagent, which creates distinct appearance of the organism against the background. Basic stains that contain positive charged chromogens are preferred since the nucleic acids of bacterial cells and cell wall components possess negative charges that strongly attracted and bonds to the anionic colorant.
• The goal of staining with simple methods is to clarify the morphology and arrangement the bacterial cells.
• The most frequently used staining agents are methylene blue crystal violet, carbol Fuchsin.
• Organisms are stained according to the hue of the stain applied.

Read Here: Simple Staining Procedure, Principle, Result

B. Negative staining

• Negative staining requires an acidic stain, such as India ink or the nigrosin. The stain that is acidic, due to it’s negatively charged chromogen is unable to penetrate the cells due to the negative charge that is present on the surface of the bacteria. Therefore, the cells that are uncolored can be easily distinguished against the background color.
• The purpose of negative staining is to investigate the morphological structure the size and arrangement of bacteria cells that are difficult to color. eg: Spirilla.
• It is also possible to colorize cells that are delicate to be heated fixed.

Read Here: Negative Staining Principle, Procedure, Result

C. Impregnation methods

Bacterial structures and cells that are too small to be observed under a light microscope, are thickened due to the impregnation with silver salts on their surface, making them apparent, e.g., for demonstration of flagella of bacterial species and Spirochetes.

1. Flagella stain (Leifson’s method)

• Flagella are too small to be seen with a bright-field microscope using normal stains, like the Gram stain or a simple stain.
• Wet mount techniques are employed to stain flagella of bacterial species It is easy and beneficial when the quantity and configuration of flagella is essential to identify the species of motile bacteria.
• The staining process requires the use of a mordant , so that the stain is able to adhere with layers on the flagella permitting visualization.
• Flagella stain Red while Vegetative cells have been stained Blue.

Read Here: Flagella Staining Principle, Procedure, Result

D. Differential staining

In this case, two staining methods are employed to impart distinct hues to various bacteria or structures of bacterial, that aid in distinguishing bacteria. The most frequently employed differential staining techniques are:

1. Gram staining

• Gram stain is an significant technique of differential staining that is used to establish the initial characterisation and classification of microbiology-related bacteria.
• Gram staining is a method to detect bacteria pathogens in the specimens and in cultures through the Gram reactions (Gram-positive as well as Gram-negative) and the morphology (cocci/rod).
• In Gram staining, Gram positive bacteria appear purple and Grams negative is pink in the color.

Read Here: Gram Staining Procedure, Principle, and Results

2. Acid fast Staining(Ziehl-Neelsen technique)

• It differentiates bacteria that are acid fast like Mycobacterium Spp. from other non-acid fast bacteria which are not stained with Gram stainin.
• In Acid-fast staining, the bacteria that stain acid-fast appears Red and Nonacid fast bacteria appear Blue in the color.

Read Here: Ziehl Neelsen Stain Principle, Procedure, Result

3. Endospore staining

• It is a way to see the structure of spores in bacteria and free spores. Only a handful of species of bacteria produce endospores, therefore the positive outcome of endospore staining methods is a significant indication of the bacterial identity. Bacillus Spp as well as Clostridium Spp constitute the primary endospore-producing bacterial genera.
• It can be used to detect Endospores within six species of bacteria.
• In Endospore staining the endospores show up as Green color, while Vegetative cells show a red.

Read Here: Endospore Staining Principle, Procedure, Result

4. Capsule staining

• Capsule stain is one type of stain that uses basic and acidic dyes to stain background and bacteria, respectively, so that capsules can be easily visible.
• It assists in proving the presence of capsules in yeasts or bacteria.
• There are two techniques for staining capsules, including Hiss Method (Positive method) and Manevals’ method (Negative).
• In Hiss method (Positive technique), the smear is stained with Hiss stain following treatment with copper sulphate.
• In Manevals’s method (Negative) the bacterial suspension smeared along with congo red and the Maneval’s stain is applied.
• Hiss stain: With this staining process the Capsule shows up in a Light violetor pale /mauve shade.
• Manevals’ method (Negative) (Negative): Purple capsule, bacterial cell is visible against a the dark background.

Read Here: Capsule Staining Procedure, Principle, Result

5. Giemsa Staining

• Giemsa stain can be described as a gold staining method that is employed for the thick and thin smears that check blood for malaria parasites and routinely check for other blood parasites, and to distinguish the morphological characteristics of the cytoplasmic and nuclear parts of Erythrocytes as well as leucocytes and Platelets and parasites.
• It is frequently employed in microbiology labs for staining:
  • Malaria and other blood parasites
  • Chlamydia trachomatis inclusion bodies
  • Borrelia species
  • Yersinia pestis
  • Histoplasma species
  • Pneumocystis jiroveci cysts (formerly Pneumocystis carinii)

Read Here: Nuclear Staining by Giemsa stain: Procedure, Principle, Result

6. Acridine Orange Staining

• This staining technique is used to determine that there are bacteria present in blood culture in cases where results from Gram staining are difficult to interpret, or in cases where the existence of bacteria has been strongly suspected, but there is no evidence to prove it with light microscopy. Acridine orange binds with nucleic acids and stain the nucleic acid molecules.
• It can also be used for the identification of Mycoplasmas (cell wall not functioning bacteria).

Read Here: Acridine Orange Staining

7. Dyar’s method for Cell wall Staining

• The most frequently used method to stain bacterial cell walls using light microscopy, is Dyar’s technique (1947) that employs the cationic surface-active agents.
• The primary purpose behind staining cells’ walls is to determine the cell wall that is populated by bacteria and to study the morphology of the cell wall.

8. Feulgen technique for Nuclear material

• Feulgen stain is one staining procedure developed in the laboratory of Robert Feulgen and is used in histology for identifying DNA or chromosomal material within cells. It is stained darkly.
• It is based on an acid-based hydrolysis reaction of DNA consequently fixing agents that use acidic substances must be avoided.
• Its Nuclear material appears to be pinkish-purple and Cytoplasm appears colorless.

9. Alberts’s method for Metachromatic granules

• Albert staining techniques aim at finding metachromatic granulated bodies in Corynebacterium diphtheriae.
• This staining technique makes the Granules are Bluish black, while Cytoplasm is seen as Green.

10. Burdon’s method for Intracellular lipids 

• This technique can be used to identify that lipids exist within the cell membrane, cell wall as well as fat globules (PHB within the cytoplasm).
• The Lipid crystals appear in Deep blue, and the Cytoplasms are in Light pink.

11. Hotch kuss method for Polysaccharide

• In this process, the polysaccharide is then oxidized using periodate to create polyaldehyde. It is able to react with Schiff’s reagents and produces a red, and cytoplasm is counter-stained using malachite’s green.
• This technique is used to identify the presence of polysaccharide granules within the cells.
• In this manner, the polysaccharide is visible in Red color, while the Cytoplasm appears in Green color.

12. Dornor’s method for Endospore Staining

• In dornor’s method stain Malachite green heat that is fixed to penetrate spores. Vegetative cells are counterstained using Safranin.
• This technique can be employed to identify that there is an endospore within six species of bacteria.
• This method makes it is observed that the Endospores show up as Green color, whereas the Vegetative cells show in Red color.

Staining Technique	Preparation	Application	Result
Simple (Monochrome)	Smear stain with single dye .eg. Methylene blue , Safranin etc	Used to highlight microbes and illustrate cellularshapes and arrangements .	Organisms are stained in the color of applied stain
Negative (Relief)	Smear mixed with Nigrosin and spreadinto thin film	Study cell morphology	Organism is stained, the background is black
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 colorGrams negative appears pink in color
Acid fast (Ziehl-Neelsen technique)	Film stained with hot Z.N.C.F. decolourized (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:RedNon acid fast: Blue
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: GreenVegetative cells: Red
CapsuleA: Hiss method (Positive technique)B: Manevals’s technique (Negative)	Smear stained with Hiss stain following treatment with copper sulphateBacterial 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 colorBacteria: Purple capsule, bacterial cell, Stands out against dark background
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
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
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
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
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
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: RedCytoplasm: Green

E. Other staining methods

1. Auramine-Rhodamine technique

• This method of staining using fluorochrome can be used to improve the identification of mycobacteria in the patient’s specimens as well as for the first characterizing cells that have been grown in a culture.

2. Calcofluor White Staining

• It is used to identify fungal components and to study the subtle traits of fungi in the laboratory.
• The cell walls of fungi be able to bind the stain calcofluor that greatly increases your ability to see fungal components in the tissue or other specimens.

3. Lactophenol cotton blue (LPCB) wet mount

• A lactophenol-coated blue (LPCB) wet mount preparation is the most commonly used method for staining and studying the growth of fungi. It is easy to prepare.

Read Here: Lactophenol Cotton Blue Staining Principle, Procedure, Result.

4. Haematoxylin and eosin (H&E) staining

• Haematoxylin and Eosin staining is often used in histology to study thin sections of tissue.
• Haematoxylin dyes cell nuclei blue, while eosin marks connective tissue, cytoplasm and extracellular compounds either red or pink.
• Eosin is extensively absorbed into red blood cells and makes the cells bright red.
• In a well-crafted H&E preparation, the red blood cells are nearly orange. Collagen and the cytoplasm (especially muscles) change to various colors of pink.

5. Papanicolaou staining

• Papanicolaou staining, also known as PAP staining, was created in order to substitute fine needle aspiration (FNAC) with the intention of reducing staining time and cost , without sacrificing quality.
• PAP staining was subject to various modifications in order to make it an “suitable alternative” for FNAC. The change was triggered by the recognition of smears that were fixed with wet by researchers who preserved the structure of the nuclei as opposed to the translucent appearance of the dry Romanowsky smears. This led to the development an hybrid stain consisting of air dried and wet fixed called Papanicolaou’s ultrafast stain.
• This modification consists of an application of the nasal solution in order to rehydrate cells, thereby increasing transparency of cells and is coupled using alcohol formalin to improve the colors that emanate from the nuclei.
• The papanicolaou stain is currently being used instead of cytological staining for every organ type because of its improvement in the quality of its morphological appearance, decreased staining timeand cost.
• It is commonly employed to stain Pap Smear specimens.
• It makes use of a mixture of haematoxylin Orange G, eosin Y, Light Green SF yellowish and, occasionally Bismarck Brown Y.

6. PAS staining

• Periodic acid-Schiff can be described as a histology special stain used to distinguish carbohydrates (glycogen glycoprotein, proteoglycans, glycoprotein).
• PAS is typically used on the liver where glycogen deposits are created. This is used to identify different types of glycogen storage disease.
• PAS is crucial as it detects glycogen granules that are found in cancers of the ovaries as well as the pancreas, which are part of the system that regulates hormone and also in kidneys and bladders of the renal system.
• Basement membranes may also appear as a stain of PAS, and could be crucial in diagnosing kidney disease.
• Due to the abundance of carbohydrate in cells of the hyphae, as well as yeast-based fungi This is why the Periodic acid -Schiff stain can assist in locating these species within tissues of the human body.

7. Masson’s trichrome

• Masson’s Trichrome is (as its name suggests) an staining method that uses three colors.
• The formula has been refined from the original Masson technique for various specific applications however all are well-suited for separate cells from the connective tissue.
• The majority of recipes result in the red color of keratin and muscle fibers and blue or green staining of bone and collagen and bone, pink or light red staining of the cytoplasm, and nuclei of cells that are black.

8. Silver staining

• Silver staining is the application of silver to color histologic sections.
• This type of staining is crucial for the analysis of proteins (for example , type III collagen) and DNA.
• It’s used to demonstrate the substances that are inside and outside cells.
• Silver staining can also be used in electrophoresis using temperature gradients.

9. Sudan staining

• Sudan staining uses Sudan dyes that stain sudanophilic compounds, usually such as oils.
• Sudan III Sudan IV, Oil Red O and Osmium tetroxide along with Sudan Black B are often utilized.
• Sudan staining is commonly used to assess the amount of fecal fat when diagnosing steatorrhea.

10. Wirtz-Conklin staining

• The Wirtz-Conklin stain is method specifically designed for staining real endospores using the use of the malachite green dye to act as the principal stain, and Safranin as the counterstain.
• When stained they will not fade away.
• Heat in the process of staining is an important contributor to the process. Heating opens the membrane of the spore, allowing the dye can get inside.
• The principal purpose behind this color is reveal the germination of spores of bacteria.
• In the event that the process of germinating occurring, then the spore will change in color because of malachite and the surrounding cell will be red due to the Safranin.
• This stain is also able to identify the direction of the spore inside the cell of the bacteria, whether it is terminal (at the end of the cell) or Subterminal (within cells) or central (completely within center of cell).

11. Collagen hybridizing peptide staining

• Collagen Hybridizing Peptide (CHP) staining provides an easy and direct method to stain collagens denatured of any kind (Type I II, IV etc.) regardless of whether they were damaged or degraded by chemical, mechanical, enzymatic or thermal processes.
• They function by refolding the collagen triple helix using the existing single strands of the tissue. CHPs are visible with the use of a simple microscope.