Types of stain in microbiology include simple staining, differential staining, negative staining, and special staining. These are different categories of staining techniques. Microbiological stains can also be classified on the basis of their chemical properties, such as basic dyes and acidic dyes. The number of types is not fixed, as the classification depends upon the basis used and some staining techniques may belong to more than one group.
Many unstained microbial cells have very little contrast when observed under a conventional brightfield microscope. This makes it difficult to observe their shape and cellular structures clearly. The selection of a stain depends upon the microorganism, the cellular structure or characteristic being examined, and the information required from microscopic observation.
What Is a Stain in Microbiology?
A stain in microbiology is a colouring or labelling reagent used to examine microorganisms under a microscope. These stains are used on microbial specimens such as bacteria, fungi and protozoa. Some stains colour the whole microbial cell or certain structures present within it. In negative staining, the background surrounding the cells is coloured while the cells remain unstained.
A dye is a colouring substance used in the preparation of many microbiological stains. The process of applying a stain to a specimen is known as staining. Conventional stains are examined by their visible colour under a light microscope. Fluorescent stains contain fluorescent dyes or labels which are examined using a fluorescence microscope.
Principle of Staining in Microbiology
Staining of microorganisms depends upon the affinity of the dye for the cellular components. The colour of a dye is due to the presence of a chromophore. In many dyes, the coloured ion carries either a positive or a negative charge. The attraction between the dye and the microbial cell depends upon their electrical charges and chemical properties.
Most bacterial cells have a negatively charged surface under normal staining conditions. Basic dyes contain positively charged coloured ions which bind to the negatively charged bacterial cells by electrostatic attraction. Acidic dyes have negatively charged coloured ions and are generally repelled by the bacterial surface. When the dye is retained by the cell, it is known as positive staining. In negative staining, the dye colours the surrounding background and the cells remain unstained. Some acidic dyes may also stain the cellular components under certain conditions.
In some staining methods, the cells are first fixed to the glass slide by heat or chemical treatment. This helps in attaching the cells to the slide and preserving their structure.
In differential staining, the primary stain is applied first to colour the cells. A mordant may be added to increase the retention of the primary stain. During decolorization, the primary stain is removed from certain cells while other cells retain it. The decolorized cells are then stained with a counterstain, which gives them a contrasting colour.
A primary stain is the first stain used in the staining procedure. It may or may not be a basic dye. A basic stain contains a positively charged coloured ion, whereas the term primary stain refers to the role of the dye in the staining method.
Objectives of Staining in Microbiology
The following are the objectives of staining in microbiology:
- Staining is used to increase the contrast between microorganisms and the surrounding background so that they can be seen clearly under the microscope.
- To study the shape and size of microorganisms, such as cocci, bacilli and spiral bacteria.
- The arrangement of bacterial cells in pairs, chains, clusters or other forms can be observed by staining.
- To differentiate bacteria into different groups based on their staining reactions. Gram staining is used to differentiate Gram-positive and Gram-negative bacteria.
- Certain bacterial structures such as capsules, endospores and flagella are not easily seen by ordinary staining methods. Special staining methods are used to observe these structures.
- Staining is used to detect the presence of microorganisms in clinical specimens. Acid-fast staining is used to detect acid-fast bacteria in samples such as sputum.
- Fluorescent staining methods are used to study the presence and distribution of cellular components such as nucleic acids and proteins.
- To differentiate between living and dead bacterial cells using specific viability stains. Some fluorescent dyes are used to detect cells with damaged membranes.
- Staining helps in the preliminary identification of microorganisms based on their shape, arrangement and staining characteristics.
Types of Staining Techniques in Microbiology
Staining techniques in microbiology are used to study microorganisms and their cellular structures under the microscope. These techniques are classified based on the staining reaction, the structures to be examined and the method used for microscopic observation.
The major types of staining techniques in microbiology are as follows:
- Simple staining
- Differential staining
- Negative staining
- Special or structural staining
- Fluorescent staining
These types are not completely separate from each other. Some staining methods belong to more than one group, such as capsule staining, which is also a type of negative staining.
1. Simple Staining
Simple staining is a staining technique in which a single dye is used to stain microorganisms.
- It is mainly used to study the shape, size and arrangement of bacterial cells.
- Basic dyes such as methylene blue, crystal violet and safranin are commonly used for simple staining.
- These dyes are positively charged and bind to the negatively charged components of bacterial cells.
- In positive simple staining, the bacterial cells take up the dye and appear coloured against a clear background.
- Different bacterial shapes such as cocci, bacilli and spiral forms can be observed by this method.
- The bacterial cells may be present singly or arranged in pairs, chains and clusters.
- A thin bacterial smear is usually air-dried and heat-fixed before applying the stain.
- The bacterial cells generally appear in the same colour, even when different types of bacteria are present in the sample.
- Simple staining does not differentiate bacteria based on their cell wall properties. Differential staining methods such as Gram staining are used for this purpose.
2. Differential Staining
Differential staining is a staining technique used to differentiate microorganisms based on their staining reactions.
- It is used to distinguish different groups of microorganisms based on their staining properties and cellular characteristics.
- Differential staining involves the use of more than one staining reagent. A primary stain, a decolorizing agent and usually a counterstain are used.
- Gram staining and acid-fast staining are the two commonly used differential staining methods.
- Gram staining is used to differentiate bacteria into Gram-positive and Gram-negative groups based on the properties of their cell walls.
- In Gram staining, crystal violet is used as the primary stain and Gram’s iodine as the mordant. The bacterial cells are then treated with alcohol or acetone-alcohol and counterstained with safranin.
- Gram-positive bacteria retain the crystal violet stain even after decolorization and appear purple under the microscope.
- Gram-negative bacteria lose the crystal violet stain during decolorization. These cells take up the safranin counterstain and appear pink or red.
- Gram-positive and Gram-negative are the two staining results obtained by Gram staining. These are not two separate Gram staining methods.
- Acid-fast staining is mainly used to differentiate acid-fast bacteria from non-acid-fast bacteria. It is commonly used for the detection of Mycobacterium species.
- Carbolfuchsin is used as the primary stain, acid-alcohol as the decolorizing agent and methylene blue as the counterstain in commonly used acid-fast staining methods.
- Acid-fast bacteria retain the carbolfuchsin stain even after treatment with acid-alcohol due to the presence of mycolic acids in their cell walls. These bacteria appear red or pink.
- Non-acid-fast bacteria lose the primary stain during decolorization and take up the methylene blue counterstain. They appear blue under the microscope.
- Ziehl–Neelsen and Kinyoun methods are commonly used for acid-fast staining. In the Ziehl–Neelsen method, heat is used during the application of carbolfuchsin, whereas the Kinyoun method does not require heating during primary staining.
3. Negative Staining
Negative staining is a staining technique in which the background is stained instead of the microbial cells.
- Nigrosin and India ink are commonly used for negative staining. These stains do not readily enter the microbial cells.
- The background is stained, while the cells remain unstained or lightly stained. The cells appear clear against a dark background under the microscope.
- It is used to study the shape, size and arrangement of microorganisms.
- Heat fixation is usually not required in negative staining. This helps to preserve the natural shape of the cells.
- Negative staining is also used to observe the capsules present around certain bacteria and yeasts.
- The capsule does not take up the stain and appears as a clear halo around the microbial cell.
- India ink staining is used to observe the capsule of Cryptococcus neoformans.
- In some capsule staining methods, an acidic dye is used to stain the background and a basic dye to stain the bacterial cell. The capsule remains unstained.
- Capsule staining is also included under special or structural staining, as it is used to observe a particular structure of the microbial cell.
4. Special or Structural Staining
Special or structural staining is used to observe certain structures of microorganisms that are not easily seen by ordinary staining methods. These include endospores, capsules and flagella.
The different methods of special staining are as follows:
- Endospore Staining– Endospore staining is used to detect endospores present in bacteria such as Bacillus and Clostridium. The Schaeffer–Fulton method uses malachite green as the primary stain and safranin as the counterstain. Heat is applied to allow malachite green to enter the endospores. The endospores appear green, while the vegetative cells appear red or pink after washing with water and counterstaining.
- Capsule Staining– It is used to observe the capsule present around certain bacterial and yeast cells. The capsule does not readily take up ordinary stains and appears as a clear zone around the cell. India ink and nigrosin are used in negative staining methods for the observation of capsules.
- Flagella Staining– Bacterial flagella are very thin and cannot be seen clearly under an ordinary light microscope. Mordants such as tannic acid or potassium alum are used to increase the apparent thickness of the flagella. Basic fuchsin may be used for staining. The number and arrangement of flagella can be observed by this method.
- Spirochete Staining– Spirochetes are thin, spiral-shaped bacteria. Special staining methods such as silver impregnation are used for their observation. Warthin–Starry staining is one of the methods used to demonstrate spirochetes in tissue sections.
- Some special staining methods are also included under differential staining. For example, endospore staining differentiates endospores from vegetative cells by staining them in different colours.
5. Fluorescent Staining
Fluorescent staining refers to using fluorescent dyes (known as fluorochrome) to look at microorganisms and their components.
- Fluorochromes take up certain wavelengths of light and give off longer wavelengths.
- The fluorochrome-dyed sample can be viewed using a fluorescence microscope. The microorganisms will therefore appear brightly stained against a dark background.
- A suitable light source, excitation filter and emission filter are required for viewing fluorescently-stained cells.
- Unlike ordinary brightfield staining where cells are viewed using transmitted light, fluorescent staining involves viewing the cells using light given off by the fluorescent dyes.
- Auramine O and auramine-rhodamine are used to stain acid-fast bacteria (particularly Mycobacterium). Acid-fast bacilli will appear bright yellow-green rods against a dark background.
- Acridine orange binds to DNA and RNA within microbial cells. It can be used to identify microorganisms and view nucleic acids. Depending on the staining conditions, the DNA may fluoresce green and the RNA rich areas will appear either orange or red.
- Calcofluor white can be used to identify fungi in clinical and laboratory samples. The dye binds to chitin and some other polysaccharides found in fungal cell walls.
- The fungal hyphae and yeast cells will appear blue-white/green under the fluorescence microscope. Depending on the type of filter used, skin scrapings and nail samples can also be viewed.
Classification of Stains Based on Chemical Nature
The stains used in microbiology are classified according to the electrical charge of their colouring components. These colouring components are known as chromophores.
The two main classes are basic stains and acidic stains. Some staining solutions contain a mixture of both types of dyes.
1. Basic Stains (Cationic Dyes)
Basic stains are dyes that contain positively charged colouring ions. These dyes have an affinity for negatively charged components of microbial cells.
Most bacterial cells have a negatively charged surface. The positively charged dye binds to the bacterial surface and other negatively charged components such as nucleic acids and certain proteins.
In basic staining, the bacterial cells take up the dye and appear coloured against an unstained background. This is known as positive staining.
Crystal violet, methylene blue, safranin, basic fuchsin and malachite green are some of the commonly used basic stains.
2. Acidic Stains (Anionic Dyes)
Acidic stains contain negatively charged colouring ions. These dyes are also known as anionic dyes.
The surface of most bacterial cells is negatively charged under ordinary staining conditions. The negatively charged dye is repelled by the bacterial surface and stains the surrounding background instead of the cells.
Nigrosin is commonly used for negative staining. The bacterial cells remain unstained and appear clear against a dark background.
Eosin and nigrosin are examples of acidic stains. Acidic dyes can also bind to positively charged components of cells and tissues. For example, eosin is used to stain certain proteins present in tissue sections.
3. Mixed or Romanowsky-Type Stains
Mixed stains contain both basic and acidic dye components in the same staining preparation.
Giemsa, Wright and Wright–Giemsa stains are examples of Romanowsky-type stains. These contain basic dyes such as azure B and related methylene blue dyes along with the acidic dye eosin.
The basic dye components stain structures containing negatively charged substances, while eosin has an affinity for positively charged cellular components. Different components of blood cells take up these dyes and appear in different colours.
Romanowsky stains are commonly used for the examination of blood cells and blood parasites. Giemsa staining is also used for the detection of certain microorganisms.
These mixed staining preparations were historically known as neutral stains. The term neutral stain does not represent a separate fundamental ionic class like basic or acidic dyes.
Common Stains Used in Microbiology and Their Applications
Different stains are used in microbiology for the examination of bacterial cells, fungal structures, endospores, capsules and other microorganisms. The selection of stain depends upon the type of microorganism and the purpose of microscopic examination.
The following are some of the commonly used stains in microbiology and their applications:
| Stain or Method | Staining Category | Primary Microbial Target | Typical Observation or Result |
|---|---|---|---|
| Methylene blue | Basic dye, simple staining | Bacterial cells | The bacterial cells appear blue. |
| Crystal violet | Basic dye, simple and Gram staining | Bacterial cells | Cells appear violet in simple staining. Gram-positive bacteria retain the violet colour in Gram staining. |
| Safranin | Basic dye, counterstaining | Gram-negative bacteria and vegetative bacterial cells | Gram-negative bacteria and vegetative cells appear pink or red after counterstaining. |
| Gram stain | Differential staining | Gram-positive and Gram-negative bacteria | Gram-positive bacteria appear purple, while Gram-negative bacteria appear pink or red. |
| Ziehl–Neelsen stain | Acid-fast staining | Acid-fast bacilli, particularly mycobacteria | Acid-fast bacilli appear red, while non-acid-fast cells usually appear blue with methylene blue counterstaining. |
| Malachite green | Basic dye, endospore staining | Bacterial endospores | Endospores appear green. Vegetative cells appear red after safranin counterstaining. |
| India ink or nigrosin | Negative staining | Bacterial and yeast capsules | The background appears dark, while the capsule is seen as a clear halo around the cell. |
| Lactophenol cotton blue | Fungal staining | Fungal hyphae, conidia and other structures | Fungal structures appear blue under the microscope. |
| Calcofluor white | Fluorescent staining | Fungal cell walls | Fungal hyphae and yeast cells show blue-white fluorescence under suitable illumination. |
| Auramine-based stains | Fluorescent acid-fast staining | Acid-fast organisms | Acid-fast bacilli show bright yellow-green or yellow-orange fluorescence, depending upon the staining preparation. |
| Giemsa stain | Romanowsky-type staining | Blood parasites and selected microorganisms | Blood parasites and their cellular structures appear in different shades of blue, purple and pink. |
Gram staining is one of the most commonly used staining methods in bacteriology. It is a differential staining method, whereas crystal violet is an individual dye used in both simple staining and Gram staining.
For the examination of fungi, lactophenol cotton blue is commonly used to observe fungal morphology. Calcofluor white is used when fluorescent examination of fungal elements is required. The staining of bacterial endospores, capsules and acid-fast organisms requires different reagents according to the structure or characteristic being examined.
The appearance of microorganisms after staining depends upon the method and reagents used. The staining result alone does not confirm the identity of a microbial species.
The quality of staining is affected by specimen preparation, fixation, thickness of the smear and condition of the staining reagents. Improper decolorization may change the staining reaction. The microscope, illumination and examination conditions also affect the observation of stained cells.
Applications of Stains in Microbiology
The following are the applications of stains in microbiology:
- Stains are used to study the shape, size and arrangement of microorganisms under the microscope.
- Gram staining is used to differentiate Gram-positive and Gram-negative bacteria based on their staining reactions.
- Detection of acid-fast bacteria such as Mycobacterium species in clinical specimens using Ziehl–Neelsen staining or fluorescent acid-fast staining.
- Endospore staining is used to detect the presence of endospores in bacteria such as Bacillus and Clostridium.
- The capsules present around certain bacterial and yeast cells can be observed using capsule staining and negative staining methods.
- Flagella staining helps in the examination of the number and arrangement of flagella in bacterial cells.
- Lactophenol cotton blue is commonly used to observe fungal structures such as hyphae, conidia and spores.
- Stains such as Giemsa are used for the detection and examination of blood parasites, including Plasmodium species, in blood smears.
- Fluorescent dyes such as acridine orange are used to detect microorganisms and examine nucleic acids present in microbial cells.
- Staining is used in clinical laboratories for the preliminary examination of microorganisms present in specimens. The staining results help in selecting further laboratory tests.
- Certain fluorescent stains such as propidium iodide are used to detect cells with damaged plasma membranes. These stains are also used in the study of microbial cell viability.
- Staining of microorganisms present in food, water and environmental samples for their microscopic examination.
Advantages of Staining in Microbiology
The following are the advantages of staining in microbiology:
- Staining increases the contrast between the microorganisms and the background. The cells can be seen more clearly under the microscope.
- The shape, size and arrangement of bacterial cells can be studied after staining.
- Gram-positive and Gram-negative bacteria can be differentiated on the basis of their staining reactions.
- It helps in the detection of microorganisms present in clinical specimens. The staining results are also used for selecting further laboratory tests.
- Detection of acid-fast bacteria such as Mycobacterium species using acid-fast staining.
- Certain bacterial structures such as capsules, endospores and flagella can be observed using special staining methods.
- Examination of fungal hyphae, spores and other structures with the help of suitable fungal stains.
- Staining is a relatively simple and rapid method for the preliminary examination of microorganisms.
- Simple staining methods require only a few reagents and commonly available laboratory equipment.
- Different types of bacterial cells present in a mixed sample can be distinguished by staining.
Limitations of Staining in Microbiology
The following are the limitations of staining in microbiology:
- In most routine staining methods, the microorganisms are killed during fixation. Their movement cannot be studied in the stained smear.
- Too much heat during fixation may change the original shape and size of the bacterial cells.
- Some microorganisms do not take up ordinary stains. These require special staining methods for their examination.
- The exact species of a microorganism cannot always be identified by staining alone. Other tests, such as biochemical and molecular tests, may be required.
- Old bacterial cultures may not give the usual Gram reaction. The result also depends upon the condition of the bacterial cells.
- The bacterial cells may overlap in a thick smear. Individual cells cannot be observed clearly.
- Improper washing may remove too much stain or leave excess stain on the smear. Excessive or insufficient decolorization may also give an incorrect Gram reaction.
- Most ordinary stains cannot differentiate between living and dead microorganisms. Special viability stains are used for this purpose.
- The concentration and quality of the staining reagents can affect the staining reaction.
- The correct interpretation of stained microorganisms requires experience in microscopic examination.
- Some staining chemicals are harmful to the skin and eyes. These require proper handling and disposal.
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