Definition: Staining is a supplementary method that gives divergence to the microscopic image for better vision under the microscope. It is a technique that is widely used for the examination of cells, tissues and cellular components.

There are a variety of staining methods like simple, differential and special staining that are used in labs to examine the bacteria. This is the primary phase in the process of identification of an organism.

This method achieved by the help of a reagent that is defined as “stain“. The process of staining can be done in two ways: in-vitro and in-vivo that is explained below:

staining 2 types
Content: Staining

  1. What is Stain
  2. Objectives
  3. Mechanism
  4. Protocol
  5. Types
  6. Examples of microorganisms
  7. Applications
  8. Conclusion

What is Stain?

It can define as a chemical reagent or dye that is responsible for the discolouration of the specimen. It adds contrast to the microscopic image that gives a distinct view of the organism.


It classifies into two types:

Based on chemical nature: It is of three kinds which are an acidic, basic and neutral stain.stain types

Based on the staining method: It is of four kinds which are direct, indirect, differential and selective stains.
staining technique

Objectives of Staining

  • Enables us to see the organism better: As microorganisms are a very minute creature as well as transparent, so it makes the specimen easy to identify.
  • Helps to differentiate organisms: Some microbes retain the colour of stain, and some don’t, which helps to distinguish between two different groups of organisms.
  • To identify a particular structure: For further study of microorganisms, it is also important to study the various internal and external structure of organism like flagella, capsule, nucleus, spores etc.


Stain generally consist of “Chromogen” and “Auxochrome”.

Chromogen: When the benzene ring reacts with the chromophore group, then the compound which is formed is called chromogen. The chromophore is a coloured compound that contains the unsaturated group, studied by O.N Witt in 1876.

Auxochrome: These are the specific groups that impart a positive and negative charge to the chromogen and is capable of ionising it. After this, the ionised stain binds to the cell with opposite charges.

Therefore, when the auxochrome group is present in the chromogen “A dye or stain is formed”.

Then this colouring agent that stains the biomolecular constituents of a specimen such as protein, as well as cellular parts that have to be dyed, depends upon the electrical charge found on the chromogen portion that imparts colour to the specimen.


Staining generally involves three steps:

  1. Preparation of smear
  2. Fixation of smear
  3. Staining of the specimen

Types of Staining

sub types of staining

Simple Staining

It determines the cell shape, size and arrangement of the microorganism. It is a very quick or simple method to perform. To perform this staining, it requires the use of a single stain only. These are of two types, namely direct and indirect staining.

Characteristics Differences Between Direct and Indirect Staining:

CharacteristicsDirect stainingIndirect staining
Stain usedBasic stainAcidic stain
Charge of stainPositiveNegative
ExamplesMethylene blue, crystal violet, carbol fuschinNigrosine, india ink, congo red
OutcomeStains the specimenStains the background
General view after stainingdirect stainingINDIRECT Staining
Principle for discolorationBecause of the positively charged stain, it gets attracted towards the negatively charged cell, hence it get fixed to the cell that retain the color of stain results in colorless background with colored cell.Because of the negatively charged stain, it gets repelled by the negatively charged cell, hence it does not fixed to the cell, results in colorless cell with colored background.

Differential Staining

It differentiates between the physical and chemical properties of two different groups of an organism based on cell-wall characteristics. To perform this staining, it requires the use of multiple or more than one stains. It is categorised into two types that are given below:

Gram staining

It identifies and classifies two major groups of bacteria, i.e. Gram-positive and Gram-negative. Dr Hans Christian Joachim Gram introduced it in 1884. This process is Carried out by differential stain known as Gram’s stain.


Gram stainingProtocolGram positive bacteriaGram negative bacteria
Primary stainingHeat fixed smear is flooded by crystal violet and allowed to stand for 1min.GRAM positiveGRAM negative
MordantingAfter washing, iodine is then flooded and allowed to stand for 1min.GRAM positiveGRAM negative
DecolourizationAfter washing, alcohol is added that is washed immediatelyGRAM positivegram negative
Counter stainingAt last, safranin is flooded over the smear and allowed to stand for 30sec, then washed by water.GRAM positivegram negative
ObservationAfter air drying, place one drop of oil immersion over the smear and adjust the microscope to identify the specimen, whether it is gram negative or gram positive.GRAM positivegram negative
Appear purple in colour because of teichoic acid that resist the primary stain.Appear pink in color due to lack of teichoic acid,alcohol creates pore in the cell which decolourizes the primary stain

Acid fast Staining

It differentiates species of mycobacterium from the other group of bacteria. Paul Ehrlich first developed it in 1882. And later, modified by Ziehl Neelson.


Acid fast stainingProtocolAcid fast bacteriaNon acid fast bacteria
Primary stainingHeat fixed smear is flooded with carbol fuschin and allowed to stand for 1 min.Acid fast non acid fast
DecolourizationAfter washing, acid alcohol is added.Acid fast non acid fast
Counter stainingAt last, methylene blue is flooded over the smear and allowed to stand for 30 sec, then wash it with waterAcid fast non acid fast
ObservationAfter air drying, place one drop of oil immersion over the smear and adjust the microscope to identify the specimen, whether specimen is acid fast or not.Acid fast non acid fast
Appears red in colour due to presence of mycolic acid that resist the color of primary stain and does not decolourize.Appears blue in colour, as they lack mycolic acid, alcohol creates pore in the cell that decolourizes the primary stain.

Special Staining

It identifies particular internal and external structural components of the specimen. It is of three types, namely Capsule, Endospore and Flagella staining.

Capsule staining

It differentiates the capsule from the rest of the cell body. This is carried out by the use of both positive and negative dyes.


Capsule acts as an envelope around the cell wall that consists of a polysaccharide.  It performs many functions like it protects the cell from desiccation, from phagocytic actions, helps in attachment of bacteria to the host cell. A capsule is responsible for the pathogenicity or virulence of an organism. This founds in both Gram-positive and Gram-negative bacteria.


Capsule stainingProtocolDiagram
Primary stainingDrop of India ink is placed on a clean slide.capsule staining
SmearingInoculum is then smeared in a dye.capsule staining
DraggingUse another slide to drag the mixture into thin film, and then air dried.capsule staining
Secondary stainingCrystal violet is flooded over the thin film, and then air dried.capsule staining
ObservationExamine the cells whether they are encapsulated or not.capsule staining
Interpretation of result
Positive: Zone formation occurs against dark background
Negative: Zone formation does not occur

Endospore Staining

It differentiates the endospore from the vegetative cell. This also makes the use of both acidic and basic stains.


As it is clear from the name endospore (endo: means inside and spore: means reproductive structure) Therefore, these are the reproductive structures that form within the cell. These are dormant spores that can resist harsh physical and chemical conditions. Mostly founds in gram-positive bacteria.

According to their position, they are of three types as given below:
endospore types


Endospore stainingProtocolDiagram
Primary stainingMalachite green is flooded over the smearendospore staining
Heat fixing Then the mixture is heat fixedendospore staining
DecolourizationDecolourized by waterendospore staining
Counter stainingSafranin is then flooded over the mixture and then air driedendospore staining
ObservationExamine the slide under the microscope, whether endospore is present or notendospore staining
Interpretation of result:
Positive: If Endospore present, it will appear green in color whereas vegetative cell appears as pink
Negative: And if endospore is absent then only vegetative cells will appear pink in color

Flagella Staining

It identifies the motility of bacteria by the presence or absence of flagella. It makes the use of acidic and neutral stain.


These are long, thread-like structures, which Protrudes outside the cell membrane. Its primary function is to provide motility or locomotion. According to the arrangement, these are of following types:

Atrichous: There are no flagella.
Monotrichous: Single flagella on one end.
Amphitrichous: Single flagella are present on both the ends.
Lophotrichous: Cluster of flagella are present on one end.
Peritrichous: Flagella are present all over the cell surface.
flagella types


Flagella stainingProtocolDiagram
Primary stainingOne drop of leifson’s stain is flooded over the smearflagella staining
Secondary stainingAfter that methylene blue is added, and allowed to stand for one minuteflagella staining
ObservationExamine the appearance of flagella to know whether the bacteria is motile or notflagella staining
Interpretation of result:
Positive: If flagella is present, then it will appear red in color while cell appears blue
Negative: And if not present, only cell will appear blue in color

Examples of bacteria in different staining method

Simple stainingDirect stain positive organism:
Staphylococcus sp. , E.coli etc
Indirect stain positive organism:
Staphylococcus sp. ,Micrococcus luteus etc
Differential stainingGram positive organisms:
Streptococcus sp. , Enterococcus sp. , Listeria sp. , Bacillus sp. etc.
Gram negative organisms:
Pseudomonas sp. , Salmonella sp. , Klebsiella sp. , Yersinia sp. etc.
Acid fast organisms:
Mycobacterium sp.
Non acid fast organisms:
Enterobacter sp.

Special stainingCapsule stain positive bacteria:
B.anthracis, K.pneumoniae etc.
Capsule stain negative bacteria:
Neisseria gonorrhoreae
Endospore stain positive bacteria:
Clostridium sp. , bacillus sp. etc.
Endospore stain negative bacteria:
E.coli , Salmonella sp. etc
Flagella stain positive organisms:
B.subtilis, pseudomonas sp. , E.coli etc
Flagella stain negative organisms:
shigella sp. , M.tuberculosis, C.diphtheriae


  • Both biological and biochemical research.
  • It is used in staining of metal.
  • Used in staining of the wood.


Various staining techniques used for different purposes like to study the morphology of bacteria, to examine internal and external cellular components. It can also be used to identify a particular group of bacteria that is necessary for further study of the specimen.

1 thought on “Staining”

  1. Elegant Akpan

    I’m grateful for adding another ideal for me pertaining to Staining and stain.
    Now that more knowledge concerning that through your website and I will like to know more.
    Thanks and God bless u.

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