Culture Media for growth and identification of microorganisms

 

Microbiology culture is a method for growing and studying microorganisms under controlled conditions in a lab. This technique allows scientists to observe microbial behavior, diagnose infections, and test the effectiveness of antibiotics for developing new treatments.

• Liquid Media: Known as Broth and it do not contain agar (the solidifying agent) this type of media allows bacteria to grow throughout the liquid.
• Solid Media: This contains agar, a gelatinous substance that provides a surface for bacteria to grow, making it easier to isolate and count colonies.

Liquid medium (Broth)

Solid medium containing Agar

Agar

Agar is derived from the cell walls of red algae, primarily from the genera Gelidium and Gracilaria. Here’s a detailed look at what agar is and its various applications.

Agar is a complex polysaccharide composed mainly of two components: agarose and agaropectin.

• Agarose is responsible for the gel-like properties of agar, forming a three-dimensional matrix that traps water.
• Agaropectin contains a mixture of sulfate esters, pyruvic acid, and other molecules that contribute to agar’s physical characteristics.

 

Agar is known for its:

• Gelation Temperature: It solidifies at around 32-40°C (90-104°F) and melts at approximately 85°C (185°F). This makes it very useful for microbiological cultures, as it remains solid at human body temperature.
• Transparency: When solidified, agar forms a clear gel, allowing for easy observation of microbial colonies.
• Non-reactivity: Agar is generally inert and does not react with most substances, making it a reliable medium for various applications.

 

Production Process

• Harvesting: Red algae are harvested from marine environments.
• Extraction: The algae are boiled to extract agar, which is then filtered to remove impurities.
• Gelation and Drying: The extracted solution is cooled to form a gel, which is then dehydrated and processed into powder, flakes, or bars.

 

Uses in Microbiology

Culture Media: Agar is most famously used as a solidifying agent in microbiological culture media mostly in Agar plates, or Petri dishes and test tubes. It provides a stable and nutrient-rich environment for microorganisms (bacteria, fungi) to grow and isolate in the form of colonies

Making Selective and differential media: Agar can be combined with various nutrients, dyes, and inhibitors to create media that select for or differentiate between specific types of microorganisms.

 

Steps in Microbiological Culture 

Sample Collection: The process starts with collecting a sample from a patient, environment, or other sources. 

Inoculation: The sample is then introduced into the culture medium by:

1. Streak Plate Method: Involves spreading a sample over an agar plate to isolate individual colonies. 
2. Pour Plate Method: A sample is mixed with molten agar at 50 degrees centigrade and pouring it into a dish to grow colonies throughout the medium.
3. Spread Plate Method: Spreading a liquid sample evenly over the surface of an agar plate for uniform growth. 

Incubation: The inoculated medium is kept at specific temperatures to promote microbial growth. 

Observation: Scientists monitor the growth, noting characteristics like colony shape, size, and color. 

Identification: Various tests, including biochemical and molecular methods, help identify the microorganisms. 

Types of Culture Media 

Different types of culture media serve specific purposes based on their composition and intended use. Here’s a detailed look at various culture media and their applications. 

1. Nutrient Media 

Example: Nutrient Agar/Broth 

Composition: Contains peptone, beef extract, and agar (for solid media). 

Use: General purpose media used to grow a wide variety of non-fastidious (no special requirements) microorganisms. It supports the growth of many types of bacteria and fungi and is commonly used for routine lab work. 

2. Selective Media 

Selective media contain substances that inhibit the growth of certain microorganisms while allowing others to grow. 

Example: MacConkey Agar 

Composition: Contains bile salts, crystal violet, lactose, and neutral red. 

Use: Selects for Gram-negative bacteria and differentiates lactose fermenters (pink colonies) from non-fermenters (colorless colonies). Commonly used to isolate Enterobacteriaceae and related Gram-negative rods from clinical specimens. 

3. Differential Media 

Differential media distinguish between different types of microorganisms based on their biological characteristics. 

Example: Blood Agar 

Composition: Nutrient agar supplemented with 5-10% sheep or horse blood. 

Use: Differentiates bacteria based on their hemolytic properties (alpha, beta, or gamma hemolysis). Useful for isolating and identifying pathogenic bacteria like Streptococcus species. 

4. Enriched Media 

Enriched media are fortified with nutrients to support the growth of fastidious organisms. 

Example: Chocolate Agar 

Composition: Heated blood agar, which releases factors V (NAD) and X (hemin) necessary for the growth of certain bacteria. 

Use: Supports the growth of fastidious organisms such as Haemophilus species and Neisseria species, which require additional growth factors not present in standard nutrient agar. 

5. Selective and Differential Media 

Some media are both selective and differential, providing a dual function. 

Example: Mannitol Salt Agar (MSA) 

Composition: High salt concentration, mannitol, and phenol red. 

Use: Selects for halotolerant bacteria like Staphylococci and differentiates based on mannitol fermentation. Mannitol fermenters (e.g., Staphylococcus aureus) produce yellow colonies due to acid production, while non-fermenters (e.g., Staphylococcus epidermidis) remain pink. 

6. Transport Media 

Transport media are used to preserve specimens and maintain the viability of organisms during transport to the laboratory. 

Example: Stuart's Transport Medium 

Composition: Contains buffers and reducing agents, lacks nutrients. 

Use: Maintains viability of microorganisms in clinical specimens without allowing them to multiply, ideal for transporting throat, wound, and urogenital swabs. 

7. Anaerobic Media 

Anaerobic media are designed to support the growth of anaerobes by creating an oxygen-free environment. 

Example: Thioglycollate Broth 

Composition: Contains thioglycollate and cystine to reduce oxygen levels. 

Use: Cultivates anaerobic bacteria by providing a low-oxygen environment. The broth gradient allows both aerobic and anaerobic bacteria to grow in different zones. 

8. Chromogenic Media 

Chromogenic media contain substrates that produce colored compounds when metabolized by specific microorganisms. 

Example: CHROMagar 

Composition: Contains chromogenic substrates specific to the enzymes of target organisms. 

Use: Rapid identification and differentiation of various bacteria and yeast species based on colony color. Commonly used in clinical microbiology to identify urinary tract pathogens and Candida species. 

9. Minimal Media 

Minimal media contain the minimum nutrients necessary for the growth of wild-type bacteria or prototrophs. 

Example: M9 Medium 

Composition: Contains glucose, salts, and minimal growth factors. 

Use: Used to culture genetically modified organisms or to study bacterial growth under nutrient-limiting conditions. Ideal for selecting bacteria with specific nutritional requirements.