Lecture 15 Flashcards
Describe reasons why cell quantitation is essential for good cell culture
measure cell performance in culture
reproducibility
comparative studies (media, serum, vessels, etc)
determine best time for sub culture
estimate plating efficiency at different densities
determine the optimum dilution
describe how hemocytometer and viability dyes work
most common method: accurate, efficient, involves calculation (total # of cells/mL = av # cells per square x dilution factor x 10^4) large squares have volume of 0.1 mm3. cells touching top and left boundary included, bottom and right are not.
trypan blue used for stains: live cells do not take up dyes while dead ones do. if exposed for too long, viable cells may take up dye
list methods for indirect and direct cell quantification
indirect: assumptions about living cells are made, actual cells are not counted. includes DNA and RNA analysis, protein analysis, and metabolic rates
direct: actual cells are counted. includes hemocytometer, resistance based electronic particle counter (Coulter/CASY), image analysis, flow cytometer, real time image analysis
explain how resistance based cell quantification (Coulter counters) work
cells with intact PM carry charges on surface. if cell is in low voltage field, no currency goes through intact membrane (insulator). if cells are aligned and sent through a small opening one after the other, live cells will change resistance that is measured.
describe image based quantification methods
programs evaluate cell images, some can discriminate between live and dead cells. can plot cell size. need to set correct thresholds, only as smart as the person setting them up!
describe principle and advantages of continuous monitoring systems
image analysis software + inverted microscope + incubator. has continues “real time” monitoring (growth curves)
explain and interpret variations between growth curves
slide 16
normal vs reduced survival vs reduced growth rate vs lower saturation density
