Differential staining Spore stain Capsule stain Spore stain

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Differential staining Spore stain Capsule stain

Differential staining Spore stain Capsule stain

Spore stain Endospores � are formed by some bacteria, aerobic Bacillus species and anaerobic

Spore stain Endospores � are formed by some bacteria, aerobic Bacillus species and anaerobic Clostridium species. � Non active, resistant to heating and chemicals. � Remain return. dormant until favorable conditions

life cycle of a spore forming bacteria

life cycle of a spore forming bacteria

� Not easily stained: � Mild heating. � Use of strong stain. � Timing

� Not easily stained: � Mild heating. � Use of strong stain. � Timing of the stain is extended.

Spore stain reagents 1 -Primary stain: Malachite green, both vegetative cell and spores will

Spore stain reagents 1 -Primary stain: Malachite green, both vegetative cell and spores will appear green. 2 -Decolorizing Agent: Water, it will remove the color from the cell, spores will remain green. 3 -Counter stain: Safranin which will be absorbed by the vegetative cell.

Spore stain steps � Apply malachite green for 5 min application of heat is

Spore stain steps � Apply malachite green for 5 min application of heat is required. � Rinse with D. water. � Stain with safranin for 1 min. � Observation: � Spores appear green While vegetative bacteria or non spore forming bacteria appear red/pink

Spore stain

Spore stain

Capsule stain � It is a negative stain where a dye does not have

Capsule stain � It is a negative stain where a dye does not have affinity for microbial cells but colors the background. � Capsule is produced by some bacteria as a slippery substance that adheres to surface forming a viscous coat. � This coat is called slime layer when it is loosely bound to bacterium.

Capsule stain � Capsules are non ionic, uncharged because they are composed of polysaccharides

Capsule stain � Capsules are non ionic, uncharged because they are composed of polysaccharides � This method use Nigrosin and Safranin to stain the bacteria and the background leaving capsule unstained as a clear halo surrounding bacteria. � Capsule is important to some bacteria (pathogens).

Capsule stain steps � Deliver 1 drop of Nigrosin by the loop and 1

Capsule stain steps � Deliver 1 drop of Nigrosin by the loop and 1 drop of safranin at the end of clean slide. � Mix a loopful of culture ( Kelbsiella sp. ) with the stain. � Spread the mixture on the slide as a blood film � Air dry the film � Leave the slide for 5 minutes at slide warmer to be fixed.

Capsule stain

Capsule stain