Hiss Staining for Demonstration of Bacterial Capsules: Introduction, Requi

Hiss Staining for Demonstration of Bacterial Capsules: Introduction, Requirements, Procedure, Result Interpretation and Significance

Introduction of Hiss Staining Technique for Demonstration of Bacterial Capsules

The Hiss Staining Technique is a type of positive staining method that stains the capsule and the bacterial cell with a brighter background. For the reason that a capsule appears as a light violet color between a dark violet-colored bacterial cell and colorless background.

Principle of Hiss Staining

The capsule is non-ionic in nature and thus it becomes a failure to get a stain by an acidic stain but the application of basic stain stains the cell as well as the capsule. Copper sulfate is a hypertonic solution that creates an ionic difference causing diffusion of the stain towards the outer surface of the cell. After drying the smear, the stain which is not passed from the capsular layer during diffusion retains in the capsular layer i.e. a dark violet color cell and light violet color capsule.

Requirements for Hiss Staining

Equipment and reagents required are-

Sprit lamp/Bunsen burner
Inoculating loop
Staining organism
Control strains ( Negative control-Non capsulated strain of E.coli while as Positive control -Capsulated strains of Klebsiella pneumoniae)
Clean and grease-free slides
Normal saline
Alcohol
1% (w/v) crystal violet
20% (w/v) copper sulfate
Microscope
Cedarwood oil

Procedure of Hiss Staining

Mix the bacterial suspension with one drop of normal saline.
Prepare dry smear by using alcohol with the help of inoculating loop.
Cover the smear with a crystal violet stain and heat gently until steam begins to rise. Leave to stain for one minute.
Wash off the stain with copper sulfate solution.
Leave the smear for drying.
Examine the smear under the oil immersion objective using cedarwood oil.

Result Interpretation of Hiss Staining

Bacterial cell: Dark purple
Capsule: Pale blue
Background: Brighter in color
Negative control (NC): Dark purple
Positive control (PC): Cell dark purple while capsule pale blue
Test organism: Either dark purple or pale blue or both

Significance of Hiss Staining

It helps to differentiate the bacteria whether capsulated or non-capsulated.
A capsule of bacteria acts as a virulence factor and gives pathogenicity to the bacterial cells.
Encapsulated bacteria are as follows-

Keynotes

Bacterial capsule observation is sometimes cumbersome since the capsule is non-ionic, so neither acidic nor basic stain and that will adhere to its surface. Another fact is most capsule materials are water-soluble, simple stains will not adhere to them.
Capsule detection is important because the capsule is a major virulence factor in the major disease-causing bacteria and thus it is essential to identify the strain.
The capsule is the outermost layer of bacteria. It, usually is composed of polysaccharides, known as capsular polysaccharides (CPS) but it may be constructed of other materials. e.g. Bacillus anthracis capsule is made of poly-D-glutamic acid. The capsule helps the organism in various ways-

Capsule assists bacteria to resist phagocytosis.
It saves the organism from desiccation.
It acts as a food reserve when certain organic compounds are in excess.
A virulence determinant of pathogenic microbes. e.g. S. pneumoniae.
The capsule also helps the organism by preventing complement-mediated bacterial cell lysis.
It protects anaerobic bacteria from oxygen toxicity.
It also excludes bacterial viruses and most hydrophobic toxic materials such as detergents.
It aids bacterial attachment to surfaces of solid objects in aquatic environments or to tissue surfaces in hosts

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