Cell Biology Lab Homework 1

This is NOT a lab group project. Do the work individually. The answers to these questions can be found in the lab protocols, appendix, or from lecture material given in class. Some questions will require outside research either in the library or on the web. 1. (10 points) Phase contrast microscopy is an important tool in cell culture work. a. Discuss how the process of phase contrast works on a microscope. b. Discuss what advantage phase contrast has over brightfield microscopy when viewing unstained cells under the microscope.

2. (25 points) Below are several practical problems that you might encounter in the cell biology laboratory. Please show all work in deriving your answers. a. How many grams of NaHCO3 (MW 84.01) are contained in 750ml of a 0.5M solution? b. You are preparing culture medium for feeding your cells. A volume of 500ml will be sufficient. Your medium needs to contain 10% fetal bovine serum. What volume of medium and serum should you add to make the 500ml of culture medium? c. For a different cell type, the medium described above must also contain 1mM glutamine. You purchase your glutamine at a concentration of 100mM. What volume of medium, serum, and glutamine need to be added to prepare the culture medium?

3. (20 points) Below are problems in cell counting. Show your work. a. The recommended volume of culture medium is between 0.2 and 0.3ml / cm2 growth area. Assume that you choose 0.3ml / cm2. What volume of medium will you need for culture flasks with a 75cm2 growth area? b. You have a cell count of 6.7 x 105 cells / ml in a total volume of 4.5ml. You have resuspended your cells in 5ml sterile PBS. You need to seed a six-well plate with the following numbers: 2 wells at 1 x 104 cells / well (2ml / well) 2 wells at 4 x 104 cells / well (2ml / well) 2 wells at 2 x 105 cells / well (2ml / well) Determine the volume of the original culture you will need to seed each concentration. 4. (15 points) The American Type Culture Collection (ATCC) is a national repository for cell lines. Researchers from all over the country send their cell lines there to be validated and shared

with others. Many of the cell lines used in the cell biology laboratory originally came from there also. Go to the ATCC website (www.ATCC.org) and search for the cell lines: Hela S3, HT-1080 and SL-29. Determine the following information if possible: a) what is the species of origin; b) what type of cell is it; c) what tissue was it isolated from; and d) what medium was it originally maintained in?

5. (30 points) Your colleague in the laboratory was able to collect all his raw data from a growth curve on the Hela S3 cell line, but had to leave unexpectedly and has asked you to perform cell calculations and prepare a growth curve to show your professor. This what your colleague did: he seeded cells into a 24-well plate (allows for replicate sampling and it is much easier to handle one 24-well plate rather than 24 flasks), and on each of the days indicated in the table on the following page, trypsinized and counted live/dead cells from three individual wells to allow for statistical accuracy. The information in the data table includes: day counted, media volume, dilutions, and number of live and dead cells counted (these numbers represent the average count from the three wells counted each day) with a Neubauer hemocytometer. You need to: a. Perform all necessary calculations to determine cell number (in this case cells/ml equals total cell number) b. Generate a growth curve by plotting cell number (on the y-axis using log scale) versus time (on the x-axis using linear scale). You need to use semi-log graph paper or Microsoft Excel (with a log y-axis). On the graph: i. ii. Label the lag, log, and plateau phases. Indicate the length of the lag phase in days on graph (by convention, lag phase is defined as the time it takes cells to exceed the original seeding concentration). Determine doubling time (doubling time for a culture is determined only using the log phase when cell growth is most homogenous). This can be determined in two ways: 1) graphically using the slope of the line. Simply pick two cell concentrations where a doubling has occurred within the log portion of the graph and determine on the x-axis how many days it took to achieve the doubling (label this process on the graph itself); 2) using the formula PDT = [ln (N/No)] / t, where PDT is the population doubling time, ln is the natural log of the number, N is the final cell number (in log phase), No is the initial cell number (in log phase), and t is the time interval between N and No.

iii.

Record of cell counts Please note that these counts have all been done with Trypan Blue. Therefore, all cultures have been diluted two-fold (equal volume of cell suspension + equal volume of Trypan Blue). Take this into account when determining cell number. Any dilution listed in table is an additional

dilution. Live and dead cells listed below represent the total number of cells in 5 large counting squares.
Live1

Day

Hemocytometer

Total Volume

Dilution

Dead

% Viability

Cells/ml (live cells only) 1 x 105

Total cell number (live cells only)* 1 x 105

0 1 2 3 4 6 8 10 12 14

Neubauer Neubauer Neubauer Neubauer Neubauer Neubauer Neubauer Neubauer Neubauer Neubauer

1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml 1ml

NA 0 0 0 0 2 5 25 25 25

NA 30 60 95 185 376 587 464 519 503

NA 2 1 1 3 5 10 12 30 80

95

*1 x 105 cells/ml cells were seeded into each well of the 24 well plate(s) on day 0 to begin the growth study. Plot
this value at the zero time point on your graph.
1

These numbers represent the total number of cells counted in 5 large squares from three separate counts.