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Special Staining; spore and capsule
Medgar Evers College
Bio 261 Microbiology
Aim #1 exercise 14
Capsule- extracellular layer composed of polysaccharides found in some bacteria that plays a role in immunity and adhesion.
It helps bacteria like
by preventing white blood cells from engulfing them and allowing them to invade the lungs.
It helps bacteria like
attach to the surface of teeth and cause dental plaque.Slide4
Problems staining the capsule
1- fixing the cells after creating a smear will destroy the delicate capsule.
2- If cells are not fixed properly, they will wash off during staining, thus you will not see anything!
We avoid this by using a combined method of negative staining and a simple stain.Slide5
1- we mix a couple of loopfuls of the
with India Ink. This will stain the background.
2- The ink suspension is spread over the slide and air dried.
3- GENTLY heat fix!
4- stain with crystal violet for one minute!
5- wash the crystal violet off with water
6- blot dry and look under microscope!Slide6Slide7
Aim #2 Exercise 16
Some species of bacteria belonging to the genera
produce tough protective structures called endospores.
Endospores are resting stages that allow the bacteria to resist harsh environmental conditions or treatments.
The spore contains a tough protein coat called the exosporium that allows it to form a barrier against all harsh conditions.
A spore is a very dehydrated structure (as much as 10 to 30 % less water than vegetative cell). The less water it has the more heat resistant.Slide8
Due to nature of the spore, it is hard to stain them and get the stain to penetrate. But, using heat as the mordant allows the primary stain to penetrate the spore.
2 methods; Schaeffer-Fulton method and Dorner methodSlide9
Malachite green is the primary stain and stains the endospore greenSafranin is the counterstain and stains the vegetative cell pink.Slide10
Only the spore is stained red with carbolfuchsin and the background is treated with Nigrosin to create contrast.