Culture Techniques Summary

Cheatsheet Content

### Plant Tissue Culture - **Definition:** 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:** - **Aseptic Technique:** Crucial to prevent contamination by microorganisms. - **Nutrient Medium:** Contains essential salts (macro/micro), vitamins, carbon source (sucrose), plant growth regulators (auxins, cytokinins). - **Environmental Control:** Temperature, light intensity, photoperiod, humidity. - **Applications:** - **Micropropagation:** Rapid clonal propagation of plants. - **Germplasm Conservation:** Storage of genetic material. - **Disease Elimination:** Production of virus-free plants (e.g., meristem culture). - **Secondary Metabolite Production:** Bioreactor culture for pharmaceuticals. - **Genetic Engineering:** Transformation and regeneration of transgenic plants. - **Steps (General):** 1. **Explant Selection:** Choose appropriate plant part (e.g., shoot tip, leaf, root). 2. **Sterilization:** Surface sterilize explant (e.g., with bleach, ethanol). 3. **Inoculation:** Place explant onto sterile culture medium. 4. **Incubation:** Grow under controlled conditions. 5. **Subculture:** Transfer to fresh medium as needed. 6. **Regeneration/Acclimatization:** Develop into whole plants and transfer to soil. ### Animal Cell Culture - **Definition:** The process of growing animal cells in an artificial environment, often outside their natural habitat. - **Key Principles:** - **Sterile Environment:** Strict aseptic technique is paramount. - **Culture Medium:** Contains essential nutrients (amino acids, vitamins, salts, glucose), growth factors (serum or serum-free alternatives), antibiotics (optional). - **Physiological Conditions:** pH (7.2-7.4), temperature ($37^\circ C$ for mammalian), $CO_2$ atmosphere (5% for bicarbonate buffering). - **Adherence vs. Suspension:** Cells can grow as monolayers (adherent) or freely floating (suspension). - **Types of Cultures:** - **Primary Culture:** Cells directly isolated from tissues. Limited lifespan. - **Cell Line:** Subcultured primary cells that can be maintained for longer periods. - **Finite/Diploid Cell Line:** Limited number of divisions (e.g., human fibroblasts). - **Continuous/Heteroploid Cell Line:** Immortal, transformed cells (e.g., HeLa, CHO). - **Applications:** - **Vaccine Production:** Growing viruses (e.g., influenza, polio) for vaccine development. - **Drug Screening:** Testing drug efficacy and toxicity. - **Monoclonal Antibody Production:** Hybridoma technology. - **Gene Therapy:** Production of viral vectors. - **Basic Research:** Studying cell biology, disease mechanisms. ### Microbial Culture: Overview - **Definition:** The process of growing microbiological organisms (e.g., bacteria, fungi, viruses) in a controlled environment. - **Key Principles:** - **Sterilization:** Media, equipment, and work area must be sterile. - **Nutrient Media:** Provides essential elements, energy source, and growth factors. Can be liquid (broth) or solid (agar plates). - **Incubation Conditions:** Optimal temperature, oxygen levels, pH. - **Aseptic Technique:** Prevents contamination of cultures. - **Types of Media:** - **Complex Media:** Composition not precisely known (e.g., LB broth, TSB). - **Defined Media:** Exact chemical composition is known. - **Selective Media:** Favors growth of specific microbes, inhibits others. - **Differential Media:** Distinguishes between different types of microbes based on characteristics. - **Techniques:** - **Streak Plating:** Isolating individual colonies from a mixed culture. - **Spread Plating:** Quantifying viable cells in a sample. - **Pour Plating:** Incorporating microbes into molten agar. - **Broth Culture:** Growing microbes in liquid medium for high cell density. ### E. coli (Escherichia coli) Culture - **Characteristics:** Gram-negative, rod-shaped bacterium, facultative anaerobe. - **Growth Media:** - **LB (Luria-Bertani) Broth/Agar:** Most common. Contains tryptone, yeast extract, NaCl. - **Minimal Media (e.g., M9):** Defined composition, used for specific metabolic studies. - **Incubation Conditions:** - **Temperature:** $37^\circ C$ (optimal). - **Aeration:** Shaking in liquid culture for aerobic growth. - **Time:** Rapid growth, doubling time ~20 minutes under optimal conditions. - **Common Techniques:** - **Transformation:** Introducing foreign DNA (plasmids) into E. coli. - **Protein Expression:** Using E. coli as a host for recombinant protein production. - **Molecular Cloning:** DNA manipulation and amplification. - **Safety:** Biosafety Level 1 (BSL-1) for non-pathogenic strains. ### Saccharomyces cerevisiae (Yeast) Culture - **Characteristics:** Unicellular eukaryote, facultative anaerobe. Model organism for eukaryotic research. - **Growth Media:** - **YPD (Yeast Extract Peptone Dextrose) Broth/Agar:** Common rich medium. - **Synthetic Defined (SD) Media:** Minimal media supplemented with specific nutrients (e.g., amino acids, bases) as needed for auxotrophic strains or selection. - **Incubation Conditions:** - **Temperature:** $30^\circ C$ (optimal). - **Aeration:** Shaking in liquid culture for aerobic respiration, static for fermentation. - **pH:** ~5-6 (slightly acidic). - **Common Techniques:** - **Transformation:** Introducing DNA (plasmids) into yeast. - **Genetic Manipulation:** Gene deletion, tagging, overexpression. - **Protein Production:** Eukaryotic protein expression. - **Fermentation:** Alcohol production. - **Safety:** Generally BSL-1. ### Influenza Virus Culture - **Characteristics:** RNA virus, obligate intracellular parasite. Requires living host cells for replication. - **Host Systems:** - **Embryonated Chicken Eggs:** Historically and still widely used for vaccine production. Virus inoculated into allantoic cavity. - **Mammalian Cell Lines:** - **MDCK (Madin-Darby Canine Kidney) cells:** Common for virus isolation, propagation, and titration. - **Vero cells:** African green monkey kidney cells, used for vaccine production. - **HEK293 cells:** Human embryonic kidney cells. - **Culture Medium:** Specific cell culture medium (e.g., DMEM, MEM) supplemented with serum (for cell growth) or serum-free (for virus production), and trypsin (for cleavage of viral hemagglutinin protein, essential for replication). - **Incubation Conditions:** - **Temperature:** $33-37^\circ C$ depending on strain and host. - **$CO_2$:** 5% for cell culture. - **Detection & Quantification:** - **Hemagglutination Assay (HA):** Detects viral particles by their ability to agglutinate red blood cells. - **Plaque Assay:** Quantifies infectious virus particles (PFU/mL). - **TCID50 (Tissue Culture Infectious Dose 50%):** Quantifies infectious dose in cell culture. - **RT-qPCR:** Detects and quantifies viral RNA. - **Safety:** Biosafety Level 2 (BSL-2) for most common strains; BSL-3 for highly pathogenic strains (e.g., H5N1).