2.5.4 Preparation of animal cell culture medium
Preparation of the culture medium is perhaps taken for granted as a simple straight-
forward procedure that is often not given due care and attention. As a result, most
infections in cell culture laboratories originate from infected media. Following the
simple yet effective procedures outlined in Section 2.4.1 should prevent or minimise
the risk of infecting the media when they are being prepared.
Preparation of the medium itself should also be carried out inside the culture
cabinet and usually involves adding a required amount of serum together with anti-
biotics to a fixed volume of medium. The amount of serum used will depend on the
cell type but usually varies between 10% and 20%. The most common antibiotics used
are penicillin and streptomycin, which inhibit a wide spectrum of Gram-positive and
Gram-negative bacteria. Penicillin acts by inhibiting the last step in bacterial cell wall
synthesis whilst streptomycin blocks protein synthesis.
Once prepared, the mixture, which is referred to as complete growth medium, should
be kept at 4C until used. To minimise wastage and risk of contamination it is advisable
to make just the required volume of medium and use this within a short period of time.
As an added precaution it is also advisable always to check the clarity of the medium
before use. Any infected medium, which will appear cloudy or turbid, should be
discarded immediately. In addition to checking the clarity, a close eye should also be
kept on the colour of the medium, which should be red at physiological pH owing to
the presence of phenol red. Media that looks acidic (yellow) or alkaline (fuchsia) should
be discarded, as these extremes will affect the viability and thus growth of the cells.2.5.5 Subculture of cells
Subculturing is the process by which cells are harvested, diluted in fresh growth
medium and replaced in a new culture flask to promote further growth. This process,
also known aspassaging, is essential if the cells are to be maintained in a healthy and
viable state, otherwise they may die after a certain period in continuous culture. The
reason for this is that adherent cells grow in a continuous layer that eventually occupies
the whole surface of the culture dish and at this point they are said to beconfluent. Once
confluent, the cells stop dividing and go into aresting statewhere they stop growing
(senesce) and eventually die. Thus, to keep cells viable and facilitate efficient trans-
formation, they must be subcultured before they reach full contact inhibition. Ideally,
cells should be harvested just before they reach a confluent state.
Cells can be harvested and subcultured using one of several techniques. The precise
method used is dependent to a large extent on whether the cells are adherent or in
suspension.Subculture of adherent cells
Adherent cells can be harvested either mechanically, using a rubber spatula (also
referred to as a ‘rubber policeman’) or enzymatically using proteolytic enzymes. Cells
in suspension are simply diluted in fresh medium by taking a given volume of cell
suspension and adding an equal volume of medium.52 Cell culture techniques