Culture Techniques

Cheatsheet Content

### Introduction to Culture Techniques Culture techniques involve growing cells, tissues, or organisms in an artificial environment under controlled conditions. These methods are crucial for research, industrial applications, and medical diagnostics. ### Plant Culture (Tissue Culture) Plant tissue culture is a collection of techniques used to maintain or grow plant cells, tissues, or organs under sterile conditions on a nutrient culture medium of known composition. #### Key Principles - **Sterility:** Absolute sterility is essential to prevent contamination by microorganisms. - **Nutrient Medium:** Contains macro/micronutrients, vitamins, amino acids, sugars, and plant growth regulators (auxins, cytokinins). - **Aseptic Technique:** All procedures are carried out in a laminar air flow hood using sterilized instruments. #### Types of Plant Culture - **Callus Culture:** Undifferentiated mass of cells grown on solid or liquid medium. Used for genetic transformation, secondary metabolite production. - **Suspension Culture:** Cells or small aggregates dispersed in a liquid medium, continuously agitated. Used for large-scale production of secondary metabolites. - **Protoplast Culture:** Plant cells without cell walls, isolated enzymatically. Used for somatic hybridization and genetic engineering. - **Embryo Culture:** Aseptic isolation and growth of immature or mature embryos. Used for overcoming seed dormancy, rescuing hybrid embryos. - **Anther/Pollen Culture:** Culturing anthers or isolated pollen grains to produce haploid plants. Useful for breeding programs and genetic studies. - **Meristem Culture:** Culturing the apical meristem to produce virus-free plants. #### Applications - Micropropagation (rapid clonal propagation of plants) - Production of disease-free plants - Genetic engineering and plant breeding - Conservation of endangered species - Production of secondary metabolites ### Animal Culture (Cell Culture) Animal cell culture involves the growth of animal cells *in vitro* in an artificial environment. #### Key Principles - **Sterility:** Crucial to prevent microbial contamination. - **Growth Medium:** Contains essential nutrients (amino acids, vitamins, salts, glucose), serum (e.g., FBS) as a source of growth factors, and antibiotics. - **Controlled Environment:** Maintenance of optimal temperature (e.g., 37°C for mammalian cells), pH (e.g., 7.2-7.4), and CO2 levels (e.g., 5% CO2 in air). - **Aseptic Technique:** Carried out in a laminar flow hood. #### Types of Animal Culture - **Primary Culture:** Cells directly isolated from tissues and grown *in vitro*. Finite lifespan. - **Cell Line:** Cells that have undergone a genetic transformation, allowing them to be subcultured indefinitely (immortalized). - **Finite Cell Lines:** Limited number of divisions (e.g., normal diploid cells). - **Continuous Cell Lines:** Can be subcultured indefinitely (e.g., cancer cells, transformed cells). - **Adherent Culture:** Cells that attach and grow on a substrate (e.g., tissue culture treated flasks). - **Suspension Culture:** Cells grown floating in the medium, typically in bioreactors or spinner flasks. #### Applications - Vaccine production - Drug screening and development - Gene therapy - Cancer research - Toxicology testing - Production of therapeutic proteins ### Microbial Culture Microbial culture is a method of multiplying microbial organisms (like bacteria and fungi) by letting them reproduce in predetermined culture medium under controlled laboratory conditions. #### General Principles - **Sterilization:** Media, glassware, and instruments must be sterile. - **Culture Media:** - **Liquid (Broth):** For large-scale growth, biochemical tests. - **Solid (Agar):** For isolation, enumeration, and storage. - **Types:** - **Defined:** Exact chemical composition known. - **Complex:** Composition not precisely known (e.g., yeast extract, peptone). - **Selective:** Inhibits growth of unwanted microbes. - **Differential:** Distinguishes between different types of microbes. - **Incubation:** Optimal temperature, oxygen levels, and time for growth. - **Aseptic Technique:** Prevents contamination. #### E. coli Culture - **Organism:** *Escherichia coli*, a Gram-negative, rod-shaped bacterium. - **Medium:** Luria-Bertani (LB) broth or agar is common. Contains tryptone, yeast extract, NaCl. - **Conditions:** Aerobic, 37°C. - **Growth:** Rapid doubling time (20-30 min) in rich medium. - **Applications:** Recombinant protein production, genetic studies, cloning. #### Saccharomyces cerevisiae Culture - **Organism:** *Saccharomyces cerevisiae* (Baker's/Brewer's yeast), a unicellular eukaryote. - **Medium:** Yeast Peptone Dextrose (YPD) broth or agar. Contains yeast extract, peptone, dextrose. - **Conditions:** Aerobic or anaerobic, 30°C. - **Growth:** Can grow on various carbon sources. - **Applications:** Fermentation (bread, alcohol), eukaryotic genetic studies, protein expression. #### Influenza Virus Culture - **Organism:** Influenza virus, an RNA virus. Cannot replicate independently. - **Host System:** Requires living host cells for replication. - **Embryonated Chicken Eggs:** Historically and still widely used for vaccine production. Virus is inoculated into the allantoic cavity. - **Mammalian Cell Lines:** - **MDCK (Madin-Darby Canine Kidney) cells:** Common for virus isolation, propagation, and titration. - **Vero cells:** Used for some vaccine production. - **Medium:** Specific cell culture medium (e.g., DMEM, MEM) supplemented with serum, antibiotics, and often trypsin for viral cleavage. - **Conditions:** 37°C, 5% CO2. - **Detection:** Hemagglutination assay (HA), plaque assay, RT-PCR, immunofluorescence. - **Applications:** Vaccine production, antiviral drug testing, basic research on viral replication and pathogenesis.