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, which are used in labs to examine the different bacterial groups.
This method is achieved by the help of a reagent termed as “stain“. A staining method uses a wide variety of natural and synthetic stains, which adds colour to the colourless specimen. The process of staining can be done in two ways, namely in-vitro and in-vivo that is explained below:
The protocol of staining generally involves three sequential stages:
Smear preparation: It is a primary stage, which involves mixing of inoculum with a drop of sterile water until the formation of thin film over the glass slide.
Fixation of smear: It is a secondary stage, which involves heat fixing of the microbial layer formed on the glass slide.
Staining of the specimen: It is the final and important stage that adds colour to the microscopic matter. It is carried out prior to microscopic examination and biochemical tests.
In this article, we will discuss the aim, mechanism, different types and procedure of the staining technique. In addition, we will also talk over the definition and types of stains based on chemical nature and staining method.
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. Stain can be classified into the following types, depending upon its chemical nature and the type of staining methods.
Based on chemical nature: There are three kinds of stain, namely an acidic, basic and neutral, depending upon the chemical nature of the stain.
Based on the staining method: There are four kinds of stain, viz. direct, indirect, differential and selective stains.
- Enables us to see the organism better: Microorganisms are very minute creatures as well as appear transparent, so staining makes the specimen easy to identify.
- Helps to differentiate organisms: Staining helps in distinguishing between the two different groups of organisms, depending upon the colour retaining ability of the cells (some microbes retain the colour of stain, while some don’t).
- 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.
A stain generally consists of “Chromogen” and “Auxochrome” group.
Chromogen: When the benzene ring reacts with the chromophore group, a chromogen group is formed. A chromophore is a coloured compound containing unsaturated group and it was studied by O.N Witt in 1876.
Auxochrome: It is a specific group that imparts a positive or negative charge to the chromogen group and also 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 stains the biomolecular constituents of a specimen such as protein, as well as cellular parts.
Types of Staining
It determines the cell shape, size and arrangement of the microorganisms. It is a very quick or simple method to perform and it makes the use of a single stain only. These are of two types, namely direct and indirect staining.
Characteristic Differences Between Direct and Indirect Staining:
|Characteristics||Direct staining||Indirect staining|
|Stain used||Basic stain||Acidic stain|
|Charge of stain||Positive||Negative|
|Examples||Methylene blue, crystal violet, carbol fuschin||Nigrosine, india ink, congo red|
|Outcome||Stains the specimen||Stains the background|
|General view after staining|
|Principle for discoloration||Because 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.|
It differentiates between the physical and chemical properties of two different groups of an organism, depending on the cell-wall characteristics. It makes the use of multiple or more than one stains. It can be categorised into two types that are given below:
It provides an important tool to differentiate the two major groups of bacteria, i.e. gram-positive and gram-negative. Dr Hans Christian Joachim Gram introduced this method in 1884. It is carried out by the use of differential stain known as Gram’s stain.
|Gram staining||Protocol||Gram positive bacteria||Gram negative bacteria
|Primary staining||Heat fixed smear is flooded by crystal violet and allowed to stand for 1min.|
|Mordanting||After washing, iodine is then flooded and allowed to stand for 1min.|
|Decolourization||After washing, alcohol is added that is washed immediately|
|Counter staining||At last, safranin is flooded over the smear and allowed to stand for 30sec, then washed by water.|
|Observation||After 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.|
|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|
It differentiates species of mycobacterium from the other groups of bacteria. Paul Ehrlich first developed it in 1882. And later, this technique was modified by a scientist named Ziehl Neelson.
|Acid fast staining||Protocol||Acid fast bacteria||Non acid fast bacteria|
|Primary staining||Heat fixed smear is flooded with carbol fuschin and allowed to stand for 1 min.|
|Decolourization||After washing, acid alcohol is added.|
|Counter staining||At last, methylene blue is flooded over the smear and allowed to stand for 30 sec, then wash it with water|
|Observation||After 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.|
|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.|
It helps in the identification of particular internal and external structural components of the specimen. It includes capsule, endospore and flagella 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: It can define as the polysaccharide envelope, which surrounds the cell wall. Capsule performs many functions like cell protection against desiccation, phagocytic actions and also helps in cell attachment to the host. A capsule is responsible for the pathogenicity or virulence of an organism. It can be seen in the cells of the gram-positive and gram-negative bacteria.
|Primary staining||Drop of India ink is placed on a clean slide.|
|Smearing||Inoculum is then smeared in a dye.|
|Dragging||Use another slide to drag the mixture into thin film, and then air dried.|
|Secondary staining||Crystal violet is flooded over the thin film, and then air dried.|
|Observation||Examine the cells whether they are encapsulated or not.|
|Interpretation of result
Positive: Zone formation occurs against dark background
Negative: Zone formation does not occur
It differentiates the endospore from the vegetative cell and makes the use of both acidic and basic stains.
Endospore: A term itself defines its meaning, in which endo stands for inside and spore stands for a reproductive structure. Therefore, endospores are the reproductive structures inherent to the cell. It acts like a dormant spore, which can resist harsh physical and chemical conditions. Endospores are commonly found in gram-positive bacteria. According to their position, they are of three types as given below:
|Primary staining||Malachite green is flooded over the smear|
|Heat fixing||Then the mixture is heat fixed|
|Decolourization||Decolourized by water|
|Counter staining||Safranin is then flooded over the mixture and then air dried|
|Observation||Examine the slide under the microscope, whether endospore is present or not|
|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
It helps in the identification of the bacterial motility through the presence or absence of flagella. It makes the use of acidic and neutral stain.
Flagella: 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: These are without flagella.
Monotrichous: Single flagellum is present at one end.
Amphitrichous: Single flagellum is present at both the ends.
Lophotrichous: Cluster of flagella are present on one end.
Peritrichous: Flagella are present all over the cell surface.
|Primary staining||One drop of leifson’s stain is flooded over the smear|
|Secondary staining||After that methylene blue is added, and allowed to stand for one minute|
|Observation||Examine the appearance of flagella to know whether the bacteria is motile or not|
|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 Methods
|Simple staining||Direct stain positive organism:|
Staphylococcus sp. , E.coli etc
|Indirect stain positive organism:
Staphylococcus sp. ,Micrococcus luteus etc
|Differential staining||Gram 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:|
|Non acid fast organisms:
|Special staining||Capsule stain positive bacteria:|
B.anthracis, K.pneumoniae etc.
|Capsule stain negative bacteria:
|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
- Staining methods have wide applicability in both biological and biochemical research.
- It is used in staining of metal.
- Used in staining of the wood.
Various staining techniques are used for different purposes like to study bacterial morphology and to examine internal and external cellular components. It can also be used to identify the particular group of bacteria, after which we can further classify the type of specimen, based on their growth behaviour and microscopic characteristics.