Types of Stains used in Microbiology

Stains in microbiology are colouring substances that are applied to microbial cells so that these cells become clearly visible under the microscope. It is used because most microbes are transparent in their natural state and cannot be seen properly.

It is the process where dyes like crystal violet, safranin, hematoxylin, eosin or Ziehl–Neelsen stain are introduced to the specimen, and the cells take up these dyes depending on their structural characters. This is referred to as an important method for increasing contrast between the organism and the background.

It helps in differentiating organisms, for example Gram-positive bacteria retain the purple colour while Gram-negative bacteria appear pink after staining. Some stains also highlight special structures like endospores, acid-fast cells or flagella which is useful for identifying pathogens.

The history of stains began in the 19th century when dyes like carmine, hematoxylin and silver nitrate were used, and later the Gram stain was developed by Hans Christian Gram in 1882. In this stain crystal violet, iodine and safranin is used to classify bacteria according to cell wall nature.

Other important stains like Ziehl–Neelsen stain appeared in the 1890s and fluorescent staining techniques were developed later. Thus, staining is the major method used for observing, identifying and studying different microbial structures in microbiology.

Different stains react or concentrate on different areas of a tissue or cell These properties can be utilized to highlight certain areas or regions. A few of the most well-known biological staining methods can be found below. If not otherwise indicated All of these dyes can be used on tissues and cells that are fixed as well as essential dyes (suitable for use in live organisms) are indicated.

A. Common Bacterial Stains

1. Basic Stains (Positively Charged)

These are the stains which bind directly to the bacterial cell. It is because the surface of most bacteria is slightly negative and the positively charged dyes get attracted to it. The bacterial cell becomes colored after staining.

Some of the main basic stains are–

• Crystal Violet
  • It is a deep purple colored dye.
  • It is the primary stain in Gram staining and most bacteria is stained purple at the initial stage.
• Safranin
  • It is pink to red in color.
  • It is used as a counterstain in Gram stain where Gram–negative bacteria is stained pink. It is also used in endospore staining where cell body is stained pink.
• Methylene Blue
  • It is blue in color.
  • It is used as a simple stain to observe bacterial shape and arrangement. It is also used as a counterstain in acid fast staining.
• Malachite Green
  • It is green in color.
  • It is used specially for endospore staining. The dye is forced into the spore with the help of heat.
• Carbol Fuchsin
  • It is bright red in color.
  • It is the primary stain for acid–fast bacteria (like Mycobacterium). It contains phenol that help in penetrating the waxy cell wall.
• Basic Fuchsin
  • It is magenta to red in color.
  • It is similar to carbol fuchsin and used as a strong counterstain for Gram–negative organisms which are hard to visualize.

2. Acidic Stains (Negatively Charged)

These are the dyes that are repelled by the negatively charged bacterial cell. So the background is stained and the bacterial cells remain clear or bright in appearance. This is referred to as negative staining.

Some common acidic stains are–

• Nigrosin
  • It is black or dark grey in color.
  • It is used for negative staining to observe delicate bacteria like spirochetes without heat fixing because heat may distort the shape.
• India Ink
  • It is black in color.
  • It is used to observe the capsule around yeast (like Cryptococcus) or bacteria. The capsule appears as a clear halo.
• Eosin
  • It is red to pink in color.
  • It is used in some differential media like EMB agar to distinguish lactose fermenters.

3. Fluorescent Stains

These stains are used under fluorescence microscope. It is the process in which the dye emits visible light when excited by UV light and bacteria appear bright colored.

Some fluorescent stains are–

• Acridine Orange
  • It stains DNA and RNA as orange.
  • It is used to detect bacteria in blood or clinical samples.
• Auramine–Rhodamine
  • It binds to mycolic acid of acid–fast bacteria.
  • It shows bright yellow–orange bacteria against dark background especially in the detection of tuberculosis.

B. Specialized & Diagnostic Stains

1. Stains for Specific Bacteria

It is used when Gram staining does not show the organisms properly or when a particular pathogen is to be identified.

Some of the main specialized stains are–

• Albert’s Stain
  • It is used for Corynebacterium diphtheriae.
  • It stains the metachromatic (volutin) granules.
  • The bacterial cell appears light green and the granules appear blue–black. These are arranged like Chinese letters.
• Gimenez Stain
  • It is used for Rickettsia and Legionella.
  • These organisms are intracellular and hard to observe.
  • The bacteria appear red to magenta and the background appears green.
• Warthin–Starry Stain
  • It is a silver stain used for spirochetes and Helicobacter pylori.
  • These organisms are very thin and cannot be seen by normal staining.
  • The organisms appear black and the background appears yellow to brown.
• Leifson Stain
  • It is used to visualize flagella.
  • It uses tannic acid to increase the thickness of flagella.
  • The flagella appear red in color.

2. Stains for Fungi (Mycology)

These stains are used because fungal cells have thick walls that do not take basic stains easily.

• Lactophenol Cotton Blue
  • It is used for molds.
  • It stains the chitin present in the fungal cell wall.
  • The hyphae and spores appear dark blue.
• Calcofluor White
  • It binds to cellulose and chitin.
  • It is a fluorescent stain.
  • The fungi appear blue–white when observed under UV light.
• Periodic Acid–Schiff (PAS)
  • It stains glycogen and mucin in the fungal cell wall.
  • Fungal elements appear bright magenta to red in tissue sections.
• Gomori Methenamine Silver (GMS)
  • It is used specially for Pneumocystis jirovecii.
  • The fungal organisms appear black with green background.

3. Stains for Parasites

These are used for detecting parasites in blood or stool samples.

• Giemsa Stain
  • It is used for Plasmodium (malaria) and other blood parasites.
  • The parasite nucleus stains red to purple and cytoplasm stains blue.
• Trichrome Stain
  • It is used for intestinal protozoa like Giardia and Entamoeba.
  • The organisms appear blue–green with red to purple nuclei.
  • The background is green.
• Modified Acid–Fast (Kinyoun)
  • It is used for Cryptosporidium.
  • The oocysts appear bright red and the background appears blue to green.

4. Stains for Specialized Structures

• Fontana–Masson Stain
  • It detects melanin.
  • It is used for Cryptococcus neoformans and also for melanin containing skin cells.
  • The melanin appears black.
• Sudan Black B
  • It stains lipids or fat.
  • It is used to detect bacteria that store fat granules.
  • The fat granules appear black to blue.

C. Blood & Parasite Stains

1. Blood Parasite Stains (Romanowsky Group)

These stains are mixtures of eosin (acidic dye) and methylene blue or azure dyes (basic). It is the most important group for detecting blood-borne parasites like malaria.

Some of the main blood parasite stains are–

• Giemsa Stain
  • It is considered the gold standard for malaria, Babesia, Trypanosoma and Leishmania.
  • The parasite nucleus stains red to purple and the cytoplasm stains blue.
  • Red blood cells appear grayish pink.
• Wright’s Stain
  • It is used for general blood smears and also for malaria and Babesia.
  • The red blood cells appear more pink to red.
  • Parasites appear blue with red chromatin.
• Field’s Stain
  • It is used for rapid malaria detection. It is mainly used for thick films.
  • Parasite chromatin appears dark red and the cytoplasm appears blue.
  • The background is clear because cells get lysed.
• Leishman Stain
  • It is used for Leishmania and also for malaria.
  • The parasite nucleus appears ruby red and cytoplasm appears blue.
  • It shows the amastigote form of Leishmania clearly.
• Acridine Orange
  • It is a fluorescent stain used for rapid malaria screening.
  • The parasites glow bright orange to green in dark background.

2. Stool & Intestinal Parasite Stains

These stains are used to detect protozoa and helminths present in fecal samples.

• Wheatley’s Trichrome Stain
  • It is the most common stool stain and used for Entamoeba histolytica and Giardia.
  • Parasite cytoplasm appears blue–green and nuclei appear dark red to purple.
  • The background is green.
• Iron Hematoxylin Stain
  • It is used for intestinal protozoa and shows clear nuclear details.
  • Organisms appear blue–gray to black.
  • It provides high contrast for identifying cysts.
• Modified Acid–Fast (Kinyoun)
  • It is used for coccidian parasites like Cryptosporidium, Cyclospora and Isospora.
  • The oocysts stain bright pink to red and background appears blue to green.
• Modified Safranin
  • It is used for Cyclospora and Isospora.
  • The dye is heated to penetrate the thick oocyst wall.
  • They stain orange to red.

3. Tissue & Other Parasite Stains

These are used when parasites are found in body tissues instead of blood or stool.

• Pneumocystis Stain (Gomori Methenamine Silver)
  • It is used for Pneumocystis jirovecii infection in lungs.
  • The cyst wall appears black and the background is green.
  • They appear like crushed ping-pong balls.
• Hematoxylin & Eosin (H&E) Stain
  • It is used for detecting general parasites in tissues like worms and larvae.
  • The nuclei stain blue and the cytoplasm stains pink.
  • It is useful in muscle biopsy for Trichinella.

D. Fluorescent Stains

1. Stains for Acid–Fast Bacteria (TB)

These stains are more sensitive than Ziehl–Neelsen stain and are used for rapid screening of Mycobacterium species.

Auramine–Rhodamine

• It binds to mycolic acids in the cell wall of Mycobacterium tuberculosis and Mycobacterium leprae.
• The bacteria glow bright yellow–orange in a dark background.
• It allows rapid scanning of sputum samples under low magnification.

2. Stains for Fungi and Yeasts

These stains bind to carbohydrates present in fungal cell walls.

Calcofluor White

• It binds to chitin and cellulose.
• The fungi glow blue–white under UV light.
• It is used for skin scrapings, nail clippings, and also to detect Pneumocystis jirovecii.

3. Stains for Nucleic Acids (DNA/RNA)

It is used to detect genetic material and is helpful in samples where bacterial count is very low.

• Acridine Orange
  • It binds to DNA and RNA.
  • RNA–rich cells like bacteria and fungi glow bright orange to red.
  • DNA–rich cells like WBCs glow green.
  • It is used for blood cultures and CSF samples when Gram stain is difficult to interpret.
• DAPI
  • It binds strongly to A–T rich regions of DNA.
  • The nuclei and bacteria glow bright blue.
  • It is used for counting microbial cells in water samples and biofilms because it stains both live and dead cells.
• Propidium Iodide (PI)
  • It binds to DNA and gives red fluorescence.
  • It cannot enter live cells with intact membranes.
  • It is used to differentiate dead cells from live cells, usually with another stain like SYTO 9.

4. Immunofluorescence (Antibody Based Stains)

It is the process where specific antibodies tagged with fluorescent dye are used to detect a particular pathogen.

FITC (Fluorescein Isothiocyanate)

• It gives apple–green fluorescence.
• Here, the antibody specific to a pathogen is attached to FITC.
• If the organism is present, the antibody binds and the organism glows green.

Some common tests are–

– FTA–ABS: It is a confirmatory test for Treponema pallidum (Syphilis).
– DFA: It is used for Bordetella pertussis and Legionella.