ENDOSPORE STAINING

ENDOSPORE STAINING

AIM

            To differentiate and identify the bacterial Endospores and vegetative cells from given bacterial culture.

PRINCIPLE

         In adverse conditions (nutrient limitation, extreme temperatures, or dehydration), some bacteria (Clostridium sp. and Bacillus sp.) produce Endospores, metabolically inactive structures that are resistant to physical and chemical damage. Endospores allow the bacterium to survive harsh conditions by protecting the genetic material of the cells; once conditions are favorable for growth, spores germinate, and bacterial growth continues. The Endospores may be spherical, sub-spherical or oval in shape, they may differ in their location within the cell (i.e., central, terminal, or sub-terminal), and they may or may not swell the cell.

            Endospores are difficult to stain with standard staining techniques because they are impermeable to dyes typically used for staining. In 1922, Dorner published a method for staining Endospores. It employed a lengthy heating step. Shaeffer and Fulton modified Dorner’s method in 1933 to make the process faster. In basic Laboratories, the simplest endospore staining technique is the Schaeffer-Fulton technique because of its easy and it is rapid to identify the bacteria.

            Endospore staining is a Differential staining technique that is used to distinguish between the vegetative cells and the Endospores. In the Schaeffer-Fulton`s method, Malachite green dye (Primary stain) along with the use of Steamed – heat (Mordant) which softens the Endospore covering allowing penetration of the dye into the Endospore. The Malachite green dye binds to the Endospore mildly and if washed with Water (Decolorizing agent), without fixing, it easily washes away, and that’s why the application of steamed Heat is important to allow the dye to penetrate the endospore. Finally, Safranin (Counter stain) is then applied, as any cells which have been decolorized. At the end of the staining process, Vegetative cells will be Pink, and Endospores will be Dark green. 

MATERIALS REQUIRED

1. Glass slide
2. Inoculation loop
3. Microscopic slide
4. Microscope
5. Bacterial culture – Bacillus sp. or Clostridium sp.
6. 0.5 % Malachite green dye
7. Water

viii.  Safranin 

PROCEDURE

a)     Clean and dry the microscopic slide thoroughly.

b)     Prepare a smear of the culture, air dry and heat fix.

c)   Place the heat fixed bacterial slide over screened water bath and then apply the primary stain Malachite green.

d)   Allow the slide to sit over the steaming water bath for 5 minutes reapplying stain if it begins to dry out.

e)     Remove the slide from the water bath, cool and rinse the slide with water until water runs clear.

f)   Flood slide with the counter stain Safranin for 1 minute then rinse view specimen under oil immersion with a compound light microscope.

Figure – 1: Steps involved in Endospore staining (Source: microbiologyinfo.com)

OBSERVATION AND RESULTS

·       Endospores - Appeared in Green colour.

·       Vegetative cells - Appeared in Red colour.

S.No	Bacteria	Endospore Staining Characteristics
1	Clostridium septicum	Thin Gram positive bacilli with numerous ovoid or citron-shaped, sub-terminal spores are present.
2	Clostridium perfringens	Relatively large “boxcar”-shaped Gram positive bacilli are seen, spores are usually present but the demonstration of spores is frequently difficult.
3	Clostridium tetani	Gram positive bacilli with the presence of round, terminal spores.
4	Clostridium sordellii	Gram positive bacilli with clumps of free spores and bacteria distended with ovoid sub-terminal spores.
5	Clostridium tertium	Thin Gram positive bacilli with sub-terminal spores
6	Bacillus cereus	Oval spores may be observed centrally or sub-terminally and the cells are not swollen in the areas where the spore is located.
7	Bacillus anthracis	Large Gram positive bacilli, with the individual cells having squared-off or slightly concave ends. Often, the organisms are arranged in chains that have the appearance of Bamboo. The spore stain shows terminal/sub-terminal spores that do not swell the vegetative cell

Figure – 2: Green coloured endospores and Red coloured vegetative cells