STAINING SPECIFIC STRUCTURES

     Many special staining procedures have been developed to study specific structures with the light microscope. They are Capsule staining, Negative staining, Endospore staining & Flagellar staining

  Capsule staining

Some bacteria have a layer of material lying outside the cell wall. When the layer is well organized and not easily washed off, it is called a capsule or glycocalyx. A glycocalyx is a network of polysaccharides/polypeptides extending from the surface of bacteria. Capsules help bacteria resist phagocytosis by host phagocytic cells. The glycocalyx also aids bacterial attachment to surfaces of solid objects in aquatic environments or to tissue surfaces in plant.

Example: Bacillus anthracis has a capsule of poly- D- glutamic acid. Streptococcus pneumoniae, Klebsiella pneumoniae Haemophilus influenzae and Pseudomonas aeruginosa have capsules.

Capsules are clearly visible in the light microscope when negative stains or special capsule stains are employed.

 Principle:

Bacterial capsules are non-ionic, so neither acidic nor basic stains will adhere to their surfaces.  Therefore, the best way to visualize them is to stain the background using an acidic stain (e.g., Nigrosine, Congo red) and to stain the cell itself using a basic stain (e.g. Crystal violet, safranin, basic fuchsin and methylene blue).

 

There are two methods:

A. Indian ink method (Negative Staining)

B. Anthony’s stain method

 

  A. Indian ink method (Negative Staining):  

—In this method two dyes, crystal violet and Indian ink are used.

— The capsule is seen as a clear halo around the microorganism against the black background.

— The background will be dark (color of Indian ink).

— The bacterial cells will be stained purple (bacterial cells takes crystal violet-basic dyes as they are negatively charged).

Observation: The capsule (if present) will appear clear against the dark background (capsule does not take any stain).

 

 

 B. Anthony’s stain method:

—In this type of capsule staining procedure, the primary stain is crystal violet, and all parts of the cell take up the purple crystal violet stain.

— There is no mordant in the capsule staining procedure.

— A 20% copper sulfate solution serves a dual role as both the decolorizing agent and counter stain.

— It decolorizes the capsule by washing out the crystal violet, but will not decolorize the cell.

— As the copper sulfate decolorizes the capsule, it also counterstains the capsule. 

Observation:  the capsule appears as a blue halo around a purple cell.

 

 

Negative staining

 Negative staining requires the use of an acidic stain such as India ink or Nigrosin. The acidic stain, with its negatively charged chromogen, will not penetrate the cells because of the negative charge on the surface of bacteria. Therefore, the unstained cells are easily noticeable against the dark/colored background.

 Advantages:

1.     Negative staining is done without heat fixation and the cells are not subjected to distortions by chemicals or heat, so their natural size and shape can be seen.

2.     Negative staining is good for bacteria that are difficult to stain, such as delicate organisms like Spirilla. Because heat fixation is not done during the staining process, slides should be handled with care because organisms are not killed.

 Procedure:

1.     Place a small drop of Nigrosin close to one end of a clean slide.

2. Using aseptic technique, place a loopful of inoculum from the bacterial culture in the drop of nigrosin and mix.
3. Place a slide against the drop of suspended organisms at a 45° angle and allow the drop to spread along the edge of the applied slide.
4. Push the slide away from the drop of suspended organisms to form a thin smear. Air-dry.
   Note: Do not heat fix the slide.
5. Examine the slides under oil immersion.

Observation: The organism is seen unstained against the black background.  The background will be dark (color of Indian ink). Negative staining is used to visualize capsules also as clear halo around the microorganism against the black background.

   

 Endospore staining (Schaeffer-Fulton method)

Vegetative cells of certain bacteria such as Bacillus spp and Clostridium spp when subjected to environmental stresses such as nutrient deprivation, they produce metabolically inactive or dormant form-endospore. Endospores are structures which are extraordinarily resistant to environmental stresses such as heat, ultraviolet radiation, gamma radiation, chemical disinfectants, and desiccation. Example of spore forming bacteria

— Bacillus anthracis cause anthrax

 — C. botulinum and C. tetani are the causative agents of botulism and tetanus, respectively.

  The structure of spore

 

·        The spore cell wall (or core wall) is inside the cortex and surrounds the core. The core has the normal cell structures such as ribosomes and a nucleoid, but is metabolically inactive.

·        — The cortex, which may occupy as much as half the spore volume, rests beneath the spore coat. It is made of a peptidoglycan that is less cross-linked than that in vegetative cells.

·        — A spore coat lies beneath the exosporium, is composed of several protein layers, and may be fairly thick. It is impermeable and responsible for the spore’s resistance to chemicals. —often is surrounded by a thin, delicate covering called the exosporium.

 

·        Principle of Spore staining

·        — A differential staining technique (the Schaeffer-Fulton method) is used to distinguish between the vegetative cells and the endospores

·        — A primary stain (malachite green) is used to stain the endospores. Which is strong stain that can penetrate the spore coat of an endospore.

·        Endospores resist to staining, the malachite green will be forced into the endospores by heating.

·        In this technique heating acts as a mordant.

·        There is no need of using any decolorizer in this spore staining as the primary dye malachite green bind relatively weakly to the cell wall but penetrate into the spore wall.

·        If washed with water the dye come out of cell wall however not from spore wall. Water is used to decolorize the vegetative cells.

·        Malachite green dye is water-soluble and does not adhere to the cell wall vegetative cells have been disrupted by heat, because of these reasons, the malachite green rinses easily from the vegetative cells.

·         As the endospores are resistant to staining, the endospores are equally resistant to de-staining and will retain the primary dye while the vegetative cells will lose the stain.

·        — The addition of a counterstain or secondary stain (safranin) is used to stain the decolorized vegetative cells.

 Procedure

1.     Fixation of bacterial cells to the surface of the microscope slide either by heating or by using methanol

2.     Application of the primary stain: Smear covered with the solution of malachite green which is strong stain that penetrate the spore coat of endospore

3.     The slide is kept on a suitable stand and heated with steam for 5 min. (Mordant

4.     The slide washed under tap water.(Decolourising agent)

5.     The slide is counter stained with safrain for about 30 sec

6.     Then the slide washed with distilled water, dried and observe under microscope.

 Observation:

 The endospores appear green in colour and vegetative cells appear in pink/red colour

 

 

  Flagella staining:  

Most motile bacteria move by use of flagella (flagellum), threadlike appendages extending from the plasma membrane and cell wall. They are about 15 or 20 μm long. Flagella are so thin they cannot be observed directly with a bright-field microscope, but must be stained with special techniques.

Bacterial species often differ distinctively in their patterns of flagella distribution.

1.     Monotrichous bacteria: (trichous means hair) have one flagella; if it is located at an end, it is said to be a polar flagellum. eg: Vibrio cholera.

2.     Lophotrichous bacteria have a cluster of flagella at one or both ends. eg: Pseudomonas. 

3.     Amphitrichous bacteria (amphi means “on both sides”) have a single flagella at each pole. eg: Spirillum

 

Flagellar Ultrastructure  

 

The bacterial flagellum is composed of three parts.

1.Filament,

2.Basal body,

3. The hook.

(1) The filament: is a hollow and cylinder constructed of a single protein called flagellin.

It is 20-nanometer-thick hollow tube. The longest portion is the filament which extends from the cell surface to the tip. The filament ends with a capping protein.

(2) Basal body is embedded in the cell and the most complex part of a flagellum.

 (3) The Hook a short, curved segment, it links the filament to its basal body and acts as a flexible coupling. The hook is made of different protein subunits.

In E. coli and most Gram-negative bacteria, the basal body has four rings. The outer L ring and P ring associate with the lipopolysaccharide and peptidoglycan layers, respectively. The inner S ring and M ring contacts the plasma membrane.  Gram positive bacteria have only two basal body rings. An inner ring connected to the plasma membrane and An outer one probably attached to the peptidoglycan

 

 

Flagella staining (Leifson’s method)

 — The Leifson’s stain is made up of Tannic acid, Basic fuchsin stain and alcohol.

— On staining the cell with Leifson’s stain, the tannic acid get attached to the flagella and alcohol gets evaporated

— After evaporation of alcohol the thickness of flagella is increased due to deposition of tannic acid and Basic fuchsin stains the Flagella.

— After this wash the slide in a gentle stream of water and treat with 1 % methylene blue for 1 minute

 Observation  Flagella appear red in colour and bacterial cell appear blue in colour.