Cell Stainings

How to Make A Smear 
1) Sterilize the loop
2) Drip DI water on the loop in order to make a thin film
3) Add the water film on the slide
4) Sterilize the loop
5) Add bacteria to the water and spread it in a circle around a penny or quarter size
6) Sterilize the loop
7) Air dry the slide
8) Heat fix the slide three times.

Simple Staining 
The bacteria smear is stained with a single reagent and makes a distinctive color from the background.
Common stains are methylene blue, crystal violet, and carbol fuschsin.
General Steps: 
1) Make a smear of bacteria
2) Add a drop of staining agent on a smear
3) Smear it out with cover slip for 20 seconds
4) NO heat fix
5) Blot dry the slide
5) Observe in microscope.

Gram Staining 
This staining technique may also refer as differential staining, which is named after Dr. Hans Christian Gram. This staining requires four different reagents which correlated to four different steps. The purpose of this staining is to distinguish gram positive and gram negative bacteria. Gram positive bacteria have around 25- 30 layers of cell walls and gram negative bacteria have around 3-5 layers of cell wall.
General Steps:
1) Make a smear of bacteria
2) Primary Stain: Immerse the slide in the crystal violet for 1 min, then rinse it with DI water gently
3) Mordant: Flood the slide with Iodine for 1 min, then rinse it with DI water which make the smear more sticky.
4) Decolorizing agent: Drip acid alcohol and rinse with DI water in the period of 10 seconds
5) Counterstain: Immerse the slide into safranin for 1 min and rinse with DI water
6) Blot dry the slide
7) Observe in microscope

Acid-Fast Staining 
Unfortunately, gram staining does not work for some bacteria, such as the genus Mycobacterium. The acid-fast staining was developed to identify M. tuberculosis and M. leprae.
Common stains: carbol fuschsin, acid-alcohol, and methylene blue.
General Steps:
1) Make a smear of bacteria
2) Primary Stain: Flood the smear with carbol fuschin on top of the hot water beaker for 5 minutes. then rinse it with DI water gently
3) Decolorizing agent: Drip acid alcohol to rinse the smear and then rinse it with DI water
4) Counterstain: Flood the smear with methylene blue. Rinse it with water gently
5) Blot dry the slide
6) Observe in microscope

Spores Staining 
Spore staining is used for the bacteria in genera Clostridium, Desulfotomaculum, and Bacillus. These bacteria have the portion called vegetative cells which are the nutrient for the bacteria and the inactive cells (spores). Malachite green is used to stain the spores.
Common stains: malachite green, safranin.
General Steps:
1) Make a smear of bacteria
2) Primary Stain: Flood the smear with malachite green for 2 to 3 minutes on top of the hot water beaker, then rinse it with DI water
3) Decolorizing agent: Rinse it with DI water until the slide is clear
4) Counterstain: Rinse it with safranin for 30 seconds and rinse it with DI water.
5) Blot dry the slide
6) Observe in microscope