Técnicas Básicas en el Laboratorio de Microbiología. Preparación de Medios de Cultivo
Preparation of Culture Media
Understanding Culture Media
- A culture medium is a natural or artificial system that supports the growth of microorganisms, with composition varying based on the species being cultivated due to differing nutritional needs.
- Some microorganisms are less demanding and thrive in standard laboratory media, while others require specific substances like vitamins, serum, or blood for growth.
Types of Culture Media
- Natural culture media include live cells such as chicken embryos or bacteria for bacteriophages. Artificial (synthetic) media are prepared in laboratories from individual components.
- Commercially available dehydrated media can be reconstituted by adding water according to manufacturer instructions regarding composition and sterilization.
Preparing Liquid and Solid Media
- Liquid media do not contain solidifying agents; their components dissolve in distilled water. Solid media use agar, a sulfated polymer derived from gelidium algae, which solidifies below 40-45°C.
- When preparing a medium, select an appropriately sized flask or vessel. For commercial dehydrated media, follow the manufacturer's recommended quantities.
Adjusting pH and Sterilization Process
- After weighing the necessary components and adding water gradually while stirring for complete dissolution, adjust the pH if needed. Dehydrated media typically come pre-adjusted.
- For solid media preparation, agar is added last. Liquid mediums are distributed into tubes using pipettes; solid mediums require melting agar before distribution.
Autoclaving for Sterilization
- To sterilize culture media, autoclaves create high-pressure steam environments that exceed 100°C to denature proteins and eliminate microorganisms.
- Standard sterilization occurs at 121°C for 20 minutes. Autoclaves must have safety features like pressure gauges and quick-disconnect systems to ensure safe operation.
Post-Sterilization Handling
- After sterilization, allow liquid mediums to cool before pouring into Petri dishes under aseptic conditions—ideally within a laminar flow hood or near a flame.