> See also: > - [[Model Systems]] > - [[Cloning and Expression Vectors]] > - [[Cell Sorting]] > - [[Microbiology]] > - [[Antimicrobial Chemotherapy]] # Cell Cultures In vitro **cell cultures** (of mammalian cells) involve the *incubation of biologically derived material (primary/immortalized) in artificial physical and chemical environments*, outside a living organism. - Bacteria such as E. coli and yeast are typically easier to work with but not the best models when studying human disease - Animal model organisms (in vivo) often yield better results as independent cells do not always respond the same as they would within an organism. However, working with animals can take much more time (breeding/growth) and requires in-depth prerequisite knowledge of the model organism. A **pure culture** will only contain one species of organism within it, making it easier to conduct experiments on. The goal of most *pure culture techniques* is to dilute bacterial samples in order to separate individual cells which can then grow up into isolated colonies when plated ## Cell Culture Media > [[Analytical Microbiology Techniques]] ![[Pasted image 20230526151646.png|300]] Cells require nutrients and the appropriate environment to grow A flask storing adherent cells is sometimes called a *culture vessel*. There are many different types of of media available: 1. **General Purpose:** Used to maintain cultures or when trying to isolate microbes from a mixed culture - Ex: Nutrient Agar (NA), Tryptic Soy Agar (TSA), Brain Heart Infusion (BHI) (more enriching) 2. **Selective:** Use inhibitors that prevent the growth of certain microorganisms. Used in the technique of [[Restriction Cloning]] - Ex: *Phenyl Ethyl Agar (PEA)* 3. **Differential** - Allow for the growth of many microbes but differentiation can be seen by indicators that detect changes that have occurred - Ex: Dyes, reagents, blood cells, culture condition, fermentation (sugars → pH change) - Includes: *Eosin Methylene Blue (EMB), Blood Agar (BA), McConky Agar (MAC)* 4. **Combination Media** - Both selective and differential - Ex: - *MacConkey Agar (Mac)* - *Eosin Methylene Blue (EMB):* Types of Nutrient Sources: - Complex media contains nutrients that have been extracted from natural sources, so their exact contents are not quantitatively known. - Defined Media has a known chemical composition for each component ### Liquid Media **Lysogeny broth (LB)** is a nutrient-rich medium often used to grow *bacterial cells*. - Synonymous with *Luria Broth*, *Life Broth* and *Luria-Bertani Medium* > [!important] Why Agar? > No known microbes are capable of metabolizing agarose, making it a useful platform for their growth. ### Solid Media Agarose gel is a solidifying agent - Derived from seaweed - Melting point > 40C ## Culture Growth A primary cell line is a cell culture that has been separated from its original tissue through chemical or mechanical mechanisms. After the first subculture, the primary cell line becomes known as a cell line or subclone. Cell lines This adds a genotypic/phenotypic disparity to what cells are kept as the slower growing cells will likely not making it to the next culture. Because of this, primary cell cultures are generally considered more accurate representations of biological systems. **Confluence** is the term commonly used as a measure of the number of cells in a cell culture dish/flash, and refers to the coverage of the dish or the flask by the cells. - The growth of a culture can be actions to take - Passaging - Changing Media - Expansion - Seeding ### Culture Passing While immortal cell lines can theoretically be passaged an infinite amount of times, it is good practice to regenerate a cell line ### Culture Seeding