Spore staining

The Spore Staining Method-Schaeffer & Fulton's Method

AIM: To perform spore staining technique to demonstrate the spore in a given culture

Principle

A spore is a dormant form of the bacterium that allows it to survive in drastic environmental conditions. Spores have a tough outer covering made of the protein keratin and are resistant to heat and chemicals. The keratin also resists staining, so extreme measures must be taken to stain the spore. In the Schaeffer-Fulton's method, a primary stain-malachite green is forced into the spore by steaming the bacterial emulsion. Malachite green is water soluble and has a low affinity for cellular material, so vegetative cells may be decolourized with water. Vegetative cells are then counterstained with safranin. Spores may be located in the middle of the cell, at the end of the cell, or between the end and middle of the cell. Spore shape may also be of diagnostic use. Spores may be spherical or elliptical. Members of the genus Corynebacterium may exhibit club shaped swellings that might be confused with spores. Spore staining distinguishes between true spores and these structures.

Procedure:

Results

Vegetative cells: red

Spores: green

PHB staining

Polyhydroxybutyrate (PHB) granules staining

A number of the micro-organisms are capable of producing polyhydroxyalkanoates (PHA’s) as storage food reserve under unbalanced growth conditions. It is similar to of synthetic plastics like polyethylene,polypropylene etc. The advantage of using PHA’s over synthetic plastic is that PHA’s are completely mineralized into carbon dioxide and water through the action of various microorganisms. PHB are produced intracellularly by various organisms such as Bacillus megaterium, Rhizobium spp., Azotobacter spp., Pseudomonas spp., etc under physiological stress conditions. These bacteria can accumulate up to 60-80% of their weight as PHB under limiting nitrogen substrate and in the presence of an abundant source of carbon (Anderson and Dawes, 1990). The major limitation in the use of biodegradable plastic is their high cost as compared to the synthetic plastic. Rhizobium species are symbiotically associated with several leguminous plants like Pisum sativam, Glycine max, Alfa alfa etc. These are Gram negative, motile, non-endospore forming bacteria. These bacteria are generally cultured in Yeast Mannitol Agar medium. Rhizobium gives colorless gummy appearance when grown on YEMA medium supplemented with congo red. The gummy appearance is because extracellular polysaccharide production. Most importantly,they are able to accumulate a high amount of PHB intracellularly.

There are two methods to achieve PHB granules staining

I. Carbol Fuchsin staining:

Carbol fuchsin staining is performed to determine the intracellular production of PHB by the isolate.

A thin smear was stained with carbol fuchsin stain for 45 seconds.

Slide washed with water and air dries the slide

Observed at 100X magnification.

The isolates capable of producing PHB showed dark colored granules of PHB intracellularly (Aneja, 2001).

II. Sudan black B staining:

 Sudan black B staining PHB producing bacteria was further confirmed using Sudan black B staining method (Schlegel et al., 1970) with some minor modifications.

 Sudan black B stain was prepared as 0.3% solution (w/v) in 60% ethanol.

The smear was prepared on glass slides and heat fixed.

            The samples were stained for 10 min with Sudan black solution, rinsed with water

 Counter stained with 0.5% safranin for 5min.

Observed at 100X magnification.

PHB granules are dark purple granules stained with Sudan black B dye