The Principles of Clinical Cytogenetics - Extra Materials - Springer

Basic Laboratory Procedures 65
amniocytes, Giant Cell Tumor Conditioned Medium for malignancies, PANDIS for breast tumors,
etc.), whereas others are appropriate for a broad spectrum of cell types (e.g., RPMI 1640, MEM). All
culture media are balanced salt solutions with a variety of additives, including salts, glucose, and a
buffering system to maintain the proper pH. Phenol red is often used as a pH indicator in many
media. If the medium becomes too acidic, it will turn yellow, whereas medium that is too basic
becomes pink or purple.
Commercial media are available either in powder forms that must be rehydrated or as ready-to-use
aqueous solutions. Both complete and incomplete types are commercially available, but most commercial
media are incomplete. Incomplete media do not contain all of the nutrients and additives
necessary for cell growth. Incomplete culture medium must be supplemented with one or more additives
before being used for cell culture:
L-Glutamine
L-Glutamine is an amino acid essential for cell growth. L-Glutamine is unstable and breaks down
on storage to D-glutamine, a form that cannot be used by cells. L-Glutamine must therefore be stored
frozen to retain its stability, and it is optimal to add it to the culture medium just prior to use. There
are some commercially available complete media that contain L-glutamine.
Serum
Serum is essential for good cell growth. Too little does not allow for maximum cell growth, but
too much can have a detrimental effect. Fetal bovine serum (FBS) is preferred; culture medium is
generally supplemented with 10–30% FBS.
Antibiotics
Microbial inhibitors are added to culture media to retard the growth of microorganisms. This is a
stopgap measure at best and should never be relied upon to compensate for sloppy technique. Good
sterile technique is always the best defense against contamination.
Penicillin/streptomycin, kanamycin, and gentamicin are bacterial inhibitors commonly used in
tissue culture. Fungicides routinely used include nystatin and amphotericin B. Fungicides can adversely
affect cell growth and are generally only used when the potential for contamination outweighs this
potentially negative effect.
Bacterial contamination of cultures imparts a cloudy appearance to the culture medium. Fungal
contamination presents to the unaided eye as “woolly” masses in the medium; when observed under
an inverted microscope, it appears as branching hyphae. Mycoplasma and viral contamination can be
hard to detect and treat. Mycoplasma should be suspected if the background level of chromosome
breaks and rearrangements is higher than usual.
Mitotic Stimulants (Mitogens)
Some cells, particularly mature lymphocytes, do not spontaneously undergo cell division and must
be stimulated to divide by the addition of an appropriate mitogen to the cell culture.
Phytohemagglutinin (PHA) is an extract of red kidney beans that stimulates division primarily of
T-lymphocytes. Cell division starts 48 hours after the addition of PHA, with additional waves of
division at 24-hour intervals. The culture period for blood specimens is based on this knowledge. For
routine peripheral blood cultures, 72 hours is usually optimal. Blood specimens from newborns might
require a shorter culture period.
Some leukemia and lymphoma studies require stimulation of B-lymphocytes. There are a number
of B-cell mitogens available, including Epstein–Barr virus, LPS (lipopolysaccharide from Escherichia
coli), protein A, TPA (12–O-tetradecanoyl-phorbol-13-acetate) and pokeweed. A cocktail
including PHA and interleukin-2 (IL-2) has proven successful as a lymphoid mitogen for bone marrow
samples.