BACE Cell Culture Fundamentals 2 — Questions and Answers
Question 1: What is the purpose of the CO2 in a cell culture incubator?
- To provide carbon for cell metabolism
- To maintain media pH by equilibrating with bicarbonate-buffered media (Correct answer)
- To prevent oxygen toxicity to cells
- To sterilize the incubator interior
Correct answer: To maintain media pH by equilibrating with bicarbonate-buffered media
CO2 (typically 5%) dissolves in the bicarbonate-buffered media, forming carbonic acid that maintains the media pH at approximately 7.2-7.4.
Most cell culture media use a sodium bicarbonate buffer system. CO2 from the incubator atmosphere dissolves in the media to form carbonic acid, which equilibrates with bicarbonate ions. With 5% CO2, most bicarbonate-buffered media maintains pH 7.2-7.4. If cells are removed from the incubator, CO2 escapes and pH rises (media turns pink with phenol red). HEPES-buffered media can maintain pH independently of CO2.
Question 2: What does the term 'passage number' refer to in cell culture?
- The number of cells seeded per flask
- The number of times cells have been subcultured (split) since the original stock (Correct answer)
- The concentration of antibiotics in the media
- The time elapsed since cells were thawed from cryostorage
Correct answer: The number of times cells have been subcultured (split) since the original stock
Passage number tracks how many times a cell line has been subcultured; high passage numbers can result in genetic drift and altered cell behavior.
Every time cells reach confluency and are trypsinized and replated, the passage number increases by one. High-passage cells may accumulate mutations, lose tissue-specific characteristics, or change growth rates — making passage number a critical experimental variable. Primary cells have finite passage limits (Hayflick limit) and senesce after 20-80 passages depending on the cell type.
Question 3: Which supplement is added to basal cell culture media to provide essential growth factors, hormones, and attachment factors?
- Antibiotic-antimycotic solution (penicillin/streptomycin)
- Fetal bovine serum (FBS) (Correct answer)
- Sodium bicarbonate buffer
- L-glutamine
Correct answer: Fetal bovine serum (FBS)
Fetal bovine serum is the most common supplement providing growth factors, hormones, adhesion proteins, and transport proteins that support cell growth and attachment.
FBS is a complex mixture containing growth factors (IGF, PDGF, FGF), hormones (insulin, cortisol), transport proteins (albumin, transferrin), adhesion factors (fibronectin, vitronectin), and nutrients. At 5-20% concentration, it supports most mammalian cell lines. FBS lot-to-lot variation makes lot testing important for reproducible results.
Question 4: What is 'mycoplasma' and why is it a concern in cell culture?
- A type of yeast that visibly contaminates cultures with white colonies
- A small, cell wall-less bacterium that infects cultures invisibly, altering cell behavior (Correct answer)
- A virus that causes cytopathic effects visible by light microscopy
- A common chemical contaminant from plasticware manufacturing
Correct answer: A small, cell wall-less bacterium that infects cultures invisibly, altering cell behavior
Mycoplasma are tiny bacteria that lack cell walls, pass through 0.22 µm filters, are invisible by light microscopy, and alter cell metabolism, gene expression, and experimental results.
Mycoplasma contamination affects an estimated 15-35% of cell cultures worldwide. Because they lack a cell wall, they are insensitive to penicillin, pass through 0.22 µm filters, and cause no visible turbidity. Effects include altered cell growth, abnormal gene expression, cytokine induction, and complete invalidation of experimental results. Detection requires PCR, Hoechst staining, or ELISA.
Question 5: When should you use trypan blue dye when working with cell cultures?
- To stain cells for visualization under a fluorescence microscope
- To assess cell viability by distinguishing live cells (exclude dye) from dead cells (take up dye) (Correct answer)
- To buffer the cell suspension during centrifugation
- To neutralize trypsin after cell detachment
Correct answer: To assess cell viability by distinguishing live cells (exclude dye) from dead cells (take up dye)
Trypan blue is a vital dye excluded by live cells with intact membranes; dead cells with damaged membranes take up the dye and appear blue, enabling viability counting.
The trypan blue exclusion assay is the standard method for assessing cell viability before subculturing or seeding experiments. Live cells with intact plasma membranes actively exclude trypan blue. Dead cells take up the blue dye and appear dark blue. Using a hemocytometer, calculate: % viability = (live cells / total cells) x 100. A viability >90% is generally required for experiments. Trypan blue should not be left in contact with cells for more than 3-5 minutes.
Question 6: What is the purpose of T-flask size designation (e.g., T-25, T-75, T-175) in cell culture?
- It indicates the volume of media required in milliliters
- It indicates the total growth surface area in cm² available for cell attachment (Correct answer)
- It specifies the maximum centrifugation speed for cells grown in that flask
- It denotes the recommended passage split ratio
Correct answer: It indicates the total growth surface area in cm² available for cell attachment
The T-designation refers to the total surface area of the flask floor in cm², which determines how many cells the flask can support at confluency.
T-flask numbers indicate the growth area: T-25 = 25 cm², T-75 = 75 cm², T-175 = 175 cm². This is critical for scaling cell culture. A T-175 flask holds approximately 7x more cells at confluency than a T-25. Knowing the surface area allows you to calculate expected cell yield, seed density (cells/cm²), and media volume (typically 0.2-0.5 mL/cm²).
BACE Cell Culture Fundamentals 2
What is the purpose of the CO2 in a cell culture incubator?