STAINING, CULTURE MEDIAS AND METHODS

STAINING, CULTURE MEDIAS AND METHODS

Staining Methods of Bacteria:

1. Gram Staining: It involves the use of a primary stain (crystal violet), a decolorizer (ethanol-acetic acid), and a counterstain (safranin or fuchsine).
2. Acid Fast Staining: It involves the use of a primary stain (carbol fuchsin), a decolorizer (acetone or ethanol), and a counterstain (methyl blue or safranin).
3. Methyl Green Staining: It involves the use of a primary stain (methyl green) and a decolorizer (acetic acid).
4. Neisser Staining: It involves the use of a primary stain (methyl violet) and a decolorizer (acetic acid).

Steps of Gram Staining:

1. Apply primary stain (crystal violet) to the bacterial smear for 1 minute.
2. Decolorize with ethanol-acetic acid for 1 minute.
3. Apply counterstain (safranin or fuchsine) for 1 minute.
4. Wash with water and dry.
5. Observe under microscope.

Gram Staining Properties:

1. Gram Positive: Bacteria that retain the primary stain and appear purple under the microscope.
2. Gram Negative: Bacteria that lose the primary stain and appear pink or red under the microscope.
3. Acid Fast: Bacteria that resist decolorization with acid and appear red under the microscope.

Differences between Gram Positive and Gram Negative Bacteria:

1. Cell Wall: Gram positive bacteria have a thick peptidoglycan cell wall, while gram negative bacteria have a thinner peptidoglycan cell wall.
2. Lipopolysaccharide: Gram negative bacteria have a lipopolysaccharide outer membrane, while gram positive bacteria do not.
3. Antigenic Properties: Gram negative bacteria have more antigenic properties than gram positive bacteria.

Steps of Acid Fast Staining:

1. Apply primary stain (carbol fuchsin) to the bacterial smear for 10-15 minutes.
2. Decolorize with acetone or ethanol for 10-15 minutes.
3. Apply counterstain (methyl blue or safranin) for 10-15 minutes.
4. Wash with water and dry.
5. Observe under microscope.

Types of Culture Media:

1. Solid Media: Media that solidifies at room temperature, such as agar or gelatin.
2. Liquid Media: Media that remains in a liquid state, such as broth or serum.
3. Semi-Solid Media: Media that is partially solidified, such as agar plate with a liquid overlay.

Examples of Culture Media:

1. Nutrient Agar: A solid medium containing peptone, meat extract, and agar.
2. Blood Agar: A solid medium containing agar, blood, and nutrients.
3. MacConkey Agar: A selective medium containing lactose, bile salts, and crystal violet.

Culture Media Constituents:

1. Nutrients: Organic compounds such as peptone, beef extract, or yeast extract.
2. Agar: A polysaccharide that solidifies the medium.
3. pH Indicator: A compound that changes color with pH changes.

Examples of Culture Media Constituents:

1. Nutrient Broth: Contains peptone, meat extract, and water.
2. Lactose Broth: Contains lactose, peptone, and water.

Culture Media Functional Requirement:

1. Selective Media: Media that inhibits the growth of certain bacteria, such as MacConkey agar.
2. Enrichment Media: Media that promotes the growth of certain bacteria, such as blood agar.
3. Maintenance Media: Media that maintains the viability of bacteria, such as nutrient broth.

Examples of Culture Media Functional Requirement:

1. Mannitol Salt Agar: A selective medium that inhibits the growth of gram negative bacteria.
2. Sabouraud Dextrose Agar: An enrichment medium that promotes the growth of fungi.

Methods of Culturing Bacteria:

1. Direct Inoculation: Inoculating bacteria directly onto the culture medium.
2. Streak Inoculation: Inoculating bacteria onto the culture medium using a streak plate.
3. Pour Plate Method: Inoculating bacteria onto the culture medium using a pour plate.

Anaerobic Culture Methods:

1. Gas Pouch Method: Using a gas pouch to create an anaerobic environment.
2. Anaerobic Jar Method: Using an anaerobic jar to create an anaerobic environment.
3. Hungate Tubes Method: Using Hungate tubes to create an anaerobic environment.

Examples of Anaerobic Culture Methods:

1. Blood Agar in Anaerobic Jar: A medium that promotes the growth of anaerobic bacteria.
2. Thioglycollate Broth: A medium that inhibits the growth of aerobic bacteria.