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What are the 4 groups of Eukaryotic Cells?
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What is the cell theory?
- Smallest unit of life
- All organisms are made of cells
- New cells come from pre-existing cells
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What is cell culture?
The process by which prokaryotic or eukaryotic cells are grown under controlled conditions
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A homogenous population of cells derived from a single parental cell is called a ______?
clone
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What famous cell was derived from cancerous growth of Henreitta Lacks?
HeLa
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Current Cell culture techniques are used for?
- Biomedical technology
- Virology
- Genetics
- Molecular Cell Biology
- Tissue Engineering
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What are some future prospects for cell culture?
- Medicine
- Targeting diseases
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What is a Primary Culture?
Cells taken directly from a tissue to a dish
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What is a secondary culture?
- Cells taken from a primary and passed or divided in vitro
- Limited divisions (ends in apoptosis)
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What is a cell line?
Cells that have mutated and won't undergo apoptosis
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What is a transformed cell line?
A cell line that has been transformed by a tumor inducing virus or chemical
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What is a hybrid cell line (hybridoma)?
Two cell types fused together with characteristics of each
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What other disciplines is cell culture related with?
- Cell biology
- Genetic Engineering
- Protein Chemistry
- Genomics
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What are the advantages of using cell culture?
- Use of animals reduced
- Cells from one cell line are homogenous and have same growth requirements
- In-vitro manipulated is easier that in vivo
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What are the disadvantages of using cell culture?
- Cell characteristics may change over time (genotype and phenotype change)
- Cells adapt to different culture environments
- Nearly impossible to recreate the in vivo environment in cell culture
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What is the purpose of designing and designating lab a working area?
- To avoid contamination
- Protect personnel
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What are the two cell culture labs spaces?
- Sterile handling section (working, incubation, storage)
- Support service area (washing, preparing, sterilization)
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What are the three classes of microbiological safety cabinets?
- I - Open fronted, low and moderate risk agents where product protection is not critical
- II - open fronted, protects from particulate contamination, low to moderate risk
- III - totally enclosed, gas tight, glove ports. High risk pathogens
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What does an incubator do?
- Provides correct growth conditions
- Humidity
- Temperature
- CO2/O2
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What is the most common temperature for ultra low freezers and what are they used for?
- -40
- Long term storage of tissue cells, DNA, RNA, and some growth medium
- (sometimes -80 for cell culture labs)
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What does an autoclave do?
Sterilizes by killing microbes with superheated steam
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What does a centrifuge do?
Seperate samples on density gradient
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What does a spectrophotometer do?
Measures nucleic acids and protein concentration
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What is the difference between a serological pipette and a micropipette?
- Serological - mL
- Micro - uL
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What do animal and plants require in their media?
hormones and growth factors (occurs in-vivo) plus essential nutrients
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What does developing cell culture media in animals depend on?
- Origin and type of cell/tissue
- Purpose of in-vitro culturing
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What are the sources of cell culture media?
- In-house - ready made in the lab and stored as stock
- Commercial- liquid or solid and may need addition growth factors or antibiotics
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What is the purpose of developing in-vitro growth media?
- To grow cells in conditions that mimics in vivo
- nutrients for the cell
- pH and aeration
- salt concentration
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What are the general types of media?
- Serum based
- Serum free
- Animal free
- Protein free or chemically defined
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What two components are in a complete culture medium?
- Basal medium (nutrients, salts and buffers)
- Supplements (hormone, growth factors, antibiotics)
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What is balance salt solutions (BSS) required in basal media?
maintain pH and prevent osmotic imbalance
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What is the primer nutrient in mammalian cell culture?
Glucose for energy
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Why should essential amino acids be supplemented to cells grown in-vitro?
The cell doesn't produce it and it needed for growth
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What are vitamins?
nutrients required for essential metabolic reactions (growth and multiplication)
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What does the vitamin content of basal medium depend on?
The cell line for which it was designed
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Why are cells cultured in a CO2 incubator?
Growing cells don't produce enough CO2 to maintain optimal pH
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What is Hepes?
An organic buffer used to prevent pH change when culture is removed from CO2 incubator
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What are the disadvantages of using Hepes?
- Toxic to some cells at elevated levels
- Higher cost
- No nutritional value
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What is the most common pH indicator and what colors does is turn for acidic, optimal, and basic?
- Optimal - Red
- Acidic- Yellow
- Basic- Purple
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What are the 5 types of growth factors
- Fibroblast
- Epithelial
- Nerve
- Transforming
- Colony Stimulating Factors
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Why are antibiotics often used in cell culture media?
Prevent or reduce microorganism contamination
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Why aren't antibiotics recommended for routine sub-culturing and stock culture?
May mask low level of contamination
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What are the most frequently used antibiotics?
- Mixtures of penicillin and streptomycin
- Gentamycin
- Fungizone
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Why shouldn't antibiotics be used continuously?
They may develop resistant microorganisms
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What is a serum?
- Yellowish, clear liquid left over after fibrin and cells are removed from blood
- Or it is a supernatant of clotted blood
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What is a serums function?
- Promotes attachment and spreading
- Supplies micronutrients and growth factor
- protects and acts as antitoxins, antioxidants, antiproteases
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What are the sources of serum?
Fetal calf, calf, horse, and bovine
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What is serum stored at and how long is it good at this temperature?
-20 and good for at least 5 years
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What are the disadvantages of serum?
- High variability between batches
- Risk of contamination
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What are the alternatives to the use of serum?
- Serum free media
- Synthetic serum
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