Exercise 12.10 - Establishment of a Primary Culture
- Chick embryo (approximately 8 days old)
- 70% (v/v) ethanol for swabbing
- Sterile scissors, forceps and probes
- Sterile petri plates
- Phosphate buffered saline (PBS)
- Trypsin, cold sterilized in a 125 ml sterile erlenmeyer containing a magnetic stirring bar
- Minimum Essential Medium
- Fetal Calf Serum
- Clinical centrifuge with sterile capped centrifuge tubes
- Culture flasks
- Inverted phase contrast microscope (Optional)
- Candle an 8 day old egg to ensure that it is alive. This is
easily accomplished by holding the egg in front of a bright light
source; the embryo can be seen as a shadow. Circle the embryo
with a pencil.
- Place the egg in a beaker with the blunt end up, and wash
the top with a mild detergent, followed by swabbing with ethanol.
- Carefully puncture the top of the egg with the point of a
pair of sterile scissors and cut away a circle of shell, thus
exposing the underlying membrane (the chorioallantois).
- With a second pair of sterile scissors, carefully cut away
and remove the chorioallantoic membrane, exposing the embryo.
- Identify and carefully remove the embryo by the neck, using
a sterile metal hook or a bent glass rod, and place the embryo in
a 100mm petri dish containing phosphate buffered saline (PBS).
Wash several times with PBS by transferring the embryo to fresh
petri plates. After removal of all yolk and/or blood, move the
embryo to a clean dish with PBS.
- Using two sterile forceps, remove the head, limbs, and
viscera. Be sure to remove the entire limb by pulling at the
proximal end. Move the remaining tissues of the embryo to yet
another dish and wash with PBS.
- Mince the embryo finely with scissors and transfer the
minced tissue to a flask containing PBS. Allow the tissue pieces
- Remove the PBS with a sterile pipette and add 25 ml of
trypsin, a proteolytic enzyme. Stir the solution gently at 37°
C for 15-20 minutes.
- Allow the larger, undigested tissue pieces to settle and
decant the supernatant into an equal volume of Minimal Essential
Medium (MEM) + 10% Fetal Calf Serum (FCS). FCS contains protease
inhibitors which will inactivate the trypsin.
- Centrifuge the cells in MEM at 1000 rpm for 10 minutes in a
standard clinical centrifuge. Remove the supernatant and
resuspend the pellet in 25 ml of fresh MEM + 10% FCS.
- Remove 0.1 ml of the culture and determine cell
concentration and viability as directed in the previous section.
- Seed two 25 cm
plastic culture flasks containing 25 ml of MEM + 10% FCS to a
final concentration of 10
- Label and place your cultures in the tissue culture
incubator at 37° C and examine daily for cell density and
- Note any changes in the color of the media. Tissue Culture
media has a pH indicator (Phenol Red) added in order to check on
the growth of cells. The media initially is a cherry red (with
slight blue haze) and turns orange and then yellow as the cells
grow, thereby reducing the media. Should this color change occur
within 24 hours, the culture is most likely contaminated and
should be disposed of.
- Examine the cultures using an inverted phase contrast
microscope. This will allow observation of the cells without
opening or disturbing the growth.
- Make cell density determinations at 10 X magnification using
a square ocular grid, as explained in Chapter One for the
determination of area.
- Plot the cell density on a log scale vs. time of culture.
- Diagram the shape of the cells at each phase.
The cultures will develop differently than the suspension
cultures. The viable cells will grow out of the trypsinized
pieces of tissue and will remain in contact with the bottom of
the culture flask. They will continue to divide and migrate until
the entire bottom of the flask is covered with a single layer of
cells (contact inhibition and the formation of a monolayer).
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org