2 . M E T H O D S
21
may occur in some developing organs from an early embryo cultured in
vitro for 24 h or less may give more biological information than the
tissue culture of small fragments of chick heart carried on for a number
of years. The definition of "culture" must inevitably remain rather
inexact, though, in general, culture will be taken to mean something
more than mere survival.
In all cases of true culture two main requirements have to be met.
The cells or tissues must first be surrounded by an environment which
is as nearly normal as possible, or at least by one to which they can
adequately adapt themselves. Secondly, contamination by other
organisms, such as bacteria, viruses, moulds, yeasts etc. and especially
by other cells brought in with tissue extracts or body-fluids, must be
scrupulously avoided.
In recent years, antibiotics have offered a tempting solution to the
problems of bacterial contamination. All too often, however, they have
been used as a slovenly substitute for proper aseptic technique and this
indiscriminate use is to be deprecated. If a strict aseptic routine is
permitted to weaken behind a shield of antibiotics the way is opened for
the ingress of viruses and other undetected contaminants. Furthermore,
antibiotics are biologically active, by definition, and the possibility
that they affect the behaviour of the cultured cells must always remain
open until specifically disproved. In many instances, of course, proper
control experiments have established that certain concentrations
of certain antibiotics have no detectable effect on the subject of study, in
which case they can be a very useful safeguard. The ones which have
proved most useful, and on the whole safe, in Tissue Culture are
penicillin, streptomycin and chloramphenicol. The concentration used
is generally 50 /xg/ml. O f these, chloramphenicol has the broadest
anti-bacterial spectrum, it is a pure synthetic substance, and at the
above concentration probably has little effect on cell metabolism
except on the production of antibodies (Ambrose and Coons, 1963).
All methods necessarily depend on the provision of a suitable medium,
and although this problem is discussed more fully in Chapter 3, it may
be convenient to consider briefly the nature of the minimal requirements in this direction and to discuss some of the problems which arise
in providing appropriate media.
Since the pioneering work of Sidney Ringer (1880) the requirement
of all tissues for some sort of salt solution as a basic minimum has been
well recognized. Natural fluids such as plasma, serum, lymph, etc.
can of course be used and these eliminate the necessity for any artificial
salt solution, since they already provide the necessary constituents. They
are, however, complex and their composition is ill-defined; their use
therefore introduces many variables and unknowns. Thus, a basal salt
21
may occur in some developing organs from an early embryo cultured in
vitro for 24 h or less may give more biological information than the
tissue culture of small fragments of chick heart carried on for a number
of years. The definition of "culture" must inevitably remain rather
inexact, though, in general, culture will be taken to mean something
more than mere survival.
In all cases of true culture two main requirements have to be met.
The cells or tissues must first be surrounded by an environment which
is as nearly normal as possible, or at least by one to which they can
adequately adapt themselves. Secondly, contamination by other
organisms, such as bacteria, viruses, moulds, yeasts etc. and especially
by other cells brought in with tissue extracts or body-fluids, must be
scrupulously avoided.
In recent years, antibiotics have offered a tempting solution to the
problems of bacterial contamination. All too often, however, they have
been used as a slovenly substitute for proper aseptic technique and this
indiscriminate use is to be deprecated. If a strict aseptic routine is
permitted to weaken behind a shield of antibiotics the way is opened for
the ingress of viruses and other undetected contaminants. Furthermore,
antibiotics are biologically active, by definition, and the possibility
that they affect the behaviour of the cultured cells must always remain
open until specifically disproved. In many instances, of course, proper
control experiments have established that certain concentrations
of certain antibiotics have no detectable effect on the subject of study, in
which case they can be a very useful safeguard. The ones which have
proved most useful, and on the whole safe, in Tissue Culture are
penicillin, streptomycin and chloramphenicol. The concentration used
is generally 50 /xg/ml. O f these, chloramphenicol has the broadest
anti-bacterial spectrum, it is a pure synthetic substance, and at the
above concentration probably has little effect on cell metabolism
except on the production of antibodies (Ambrose and Coons, 1963).
All methods necessarily depend on the provision of a suitable medium,
and although this problem is discussed more fully in Chapter 3, it may
be convenient to consider briefly the nature of the minimal requirements in this direction and to discuss some of the problems which arise
in providing appropriate media.
Since the pioneering work of Sidney Ringer (1880) the requirement
of all tissues for some sort of salt solution as a basic minimum has been
well recognized. Natural fluids such as plasma, serum, lymph, etc.
can of course be used and these eliminate the necessity for any artificial
salt solution, since they already provide the necessary constituents. They
are, however, complex and their composition is ill-defined; their use
therefore introduces many variables and unknowns. Thus, a basal salt