1. Culture Media for growing cells

3. Animal cell. Animal cells do not have a cell wall and, thus, do not have a rigid cell boundary. The shapes of animal cells are quite diverse due to the flexibility of the outer membrane and the response when cells touch each other.
Plant cell. Most plant cells contain chloroplasts and a rigid cell wall. Animal cells do not.

4. Each rod-shaped structure in this electron micrograph is an E
Each rod-shaped structure in this electron micrograph is an E. coli cell. E. coli cells are simple prokaryotes with no membrane-bound organelles, such as mitochondria or chloroplasts.
Shown in cell culture, these CHO cells are a common mammalian cell line used to manufacture recombinant protein.

5. The Molecules of Cells
Engineered molecules are the basis of many biotechnology products.
Cells are composed of a variety of molecules.
Many molecules found in cells are much larger than atoms.
Very large molecules are found in structural components.

6. Carbohydrates
Carbon, hydrogen, and oxygen
Ratio 1:2:1
Polysaccharides
Excellent structural and energy-storing molecules
Plants store glucose in starch molecules
Monosaccharides
Monomer units that cells use to build polysaccharides
Most well known is glucose; an energy molecule

7. Disaccharides
Produced when enzymes form a bond between two monosacchrides
Sucrose is made when fructose and glucose are chemically combined
Lipids
Often referred to as hydrocarbons
Three groups of lipids
Triglycerides
Phospholipids
Steroids

8. Proteins
Nucleic Acids
The most important of the cellular molecules
Nine different categories of proteins
Structural
Enzyme
Transport
Contractile
Hormone
Antibody
Pigment
Recognition
Toxins
A typical cell produces more than 2000 proteins
Amino acids are the monomers of proteins
There are 20 different amino acids found in proteins
The fourth major group of macromolecules
Two types of nucleic acids
DNA
RNA

9. Bacterial Culture Media
Liquid vs. Solid/Semisolid
Chemically defined vs. Complex
Selective and Differential Media

10. Nutrient Requirements for Bacteria
Element
% of Dry weight
Function
Media source
Carbon 50
Main building block
Sugars, extracts
Oxygen 20
Electron acceptor, water
Air
Nitrogen 14
Amino acids, nucleic acids, coenzymes
Amino acids, extracts
Hydrogen 8
Organic compounds and water
Present in most chemicals added to media
Phosphorus 3
Nucleic acids
Na and K phosphates
Sulfur 1
Amino acids & coenzymes
Na sulfate
Minerals (Na, K, Mg, Ca, Fe etc.)
2 – 4
Transport, cofactors etc.
Salts
Trace Elements
Trace
Variety of functions
Salts of Co, Zn, Mo, Cu, Ni, Se, etc.
Organic growth factors
Growth factor
Vitamins, purines, pyrimidines

11. Sources of Nutrients Yeast Extract Peptones
Glucose (or other defined carbon sources such as acetate, glycerol, lipids etc.)
Ammonium salts (for nitrogen)
Mineral salts for trace elements (e.g. CaCl2, CuSO4 etc.)
Growth factors ( e.g. vitamins, purines, pyrimidines etc.) needed for more fastidious bacteria
Other supplements: antibiotics, pH indicators etc.

12. Method of making bacterial culture media
Mix all ingredients in purified water
Adjust pH if necessary
Sterilize by autoclaving at 121oC for 15 minutes
Cool medium to 55oC before adding sterile stock of antibiotic before pouring agar plates
Some components may need to be autoclaved separately to avoid precipitation or unwanted chemical reactions

13. Nutrient Requirements for Cultured Mammalian Cells
Component
Function
Media source
Carbohydrates & derivatives
Primary energy source
Glucose
Bulk ions and trace elements
Transport, adhesion, signaling, cofactors etc.
Salts of Ca, Cu, K, Na, Fe, Mg, Zn etc.
Amino Acids
Nitrogen source
Amino acids
Vitamins & coenzymes
Enzymatic cofactors
Vitamins (e.g. Biotin, folic acid, Vitamin B12 etc.)
Lipids and derivatives
Components of cell membrane
Lipoic and linoleic acids, inositol, choline chloride
Purines & pyrimidines
Nucleic acid precursurs
Hypoxanthine, thymidineHCl
Proteins
Growth factors, cytokines, attachment & transport
Fetal Bovine Serum (FBS) or several proteins
Non-hormonal & Non-nutritional supplements
Buffers, antibiotics, pH indicators etc.
Bicarbonate, pen/strep, phenol red

14. Media Selection: Commonly available basal media
Media Type
Common Uses
Minimum Essential Medium (MEM)
Broad spectrum
Basal Media Eagle (BME)
Diploid/ primary cells
Dulbecco’s Modified Eagle (DME)
CMRL (?)
Earl’s “L”, monkey kedney cells
Iscove’s Modified Dulbecco’s (IMDM)
Rapidly proliferating high density cultures
McCoy’s
Human lymphocytes
Ham’s F10 and F12
CHO cells
RPMI 1630/1640
Suspension cells
11/11/2018

15. Making Media for Cell Culture
Basal media is store bought
Liquid, powder, or concentrate
Add Serum, antibiotics, additional amino acids etc.
Filter sterilize with 0.2 or 0.1 micron filter into pre-sterilized bottles