Special Staining; spore and capsule

Lab #4

Medgar Evers College

Bio 261 Microbiology

Prof. Santos

Aim #1 exercise 14

• Capsular staining

• Capsule- extracellular layer composed of polysaccharides found in some bacteria that plays a role in immunity and adhesion.

• It helps bacteria like Streptococcus pneumonia by preventing white blood cells from engulfing them and allowing them to invade the lungs.

• It helps bacteria like Streptococcus mutans attach to the surface of teeth and cause dental plaque.

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.

procedure

1- we mix a couple of loopfuls of the Klebsiella pneumonia 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!

Aim #2 Exercise 16

• Spore stain

• Some species of bacteria belonging to the genera Bacillus and Clostridia 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.

• 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 method

Schaeffer- Fulton

• Malachite green is the primary stain and stains the endospore green

• Safranin is the counterstain and stains the vegetative cell pink.

Dorner method

• Only the spore is stained red with carbolfuchsin and the background is treated with Nigrosin to create contrast.