conditions that prevent contaminating microorganisms from the environment from
entering the cultures. Part of the precaution taken involves washing hands with
antiseptic soap and ensuring that all work surfaces are kept clean and sterile by
swabbing with 70% IMS before starting work. Moreover, all procedures, including
media preparation and cell handling, should be carried out in a cell culture cabinet
that is maintained in a clean and sterile condition.
Other essential precautions should include avoiding talking, sneezing or coughing
into the cabinet or over the cultures. A clean pipette should be used for each different
procedure and under no circumstance should the same pipette be used between different
bottles of media, as this will significantly increase the risk of cross-contamination.
All spillages must be cleaned quickly to avoid contamination from microorganisms
that may be present in the air. Failing to do so may result in infections to the cultures,
which may be reduced by using antibiotics. However, this is not always guaranteed
and good aseptic techniques should eliminate the need for antibiotics. In the event
of cultures becoming contaminated, these should be removed immediately from
the laboratory, disinfected and autoclaved to prevent the contamination spreading.
Under no circumstance can an infected culture be opened inside the cell culture
cabinet or incubator. Moreover, all waste generated must be decontaminated and
disposed of immediately after completing the work. This should be carried out in
accordance with the national legislative requirements, which state that cell culture
waste including media be inactivated using a disinfectant before disposal and that
all contaminated materials and waste be autoclaved before being discarded
or incinerated.
The risk from infections is the most common cause for concern in cell culture.
Various factors can contribute to this, including poor work environment, poor aseptic
techniques and indeed poor hygiene of the operator. The last of these is important,
since most of the common sources of infections such as bacteria, yeast and fungus
originate from the worker. Maintaining a clean environment and adopting good
laboratory practice and aseptic techniques should, therefore, help to reduce the risks
of infection. However, should infections occur, it is advisable to address this immedi-
ately and eradicate the problem. To do this, it helps to know the types of infection to
expect and what to look for.
In animal cell cultures, bacterial and fungal infections are relatively easy to identify
and isolate. The other most common contamination originates from mycoplasma.
These are the smallest (approximately 0.3 mm in diameter) self-replicating prokary-
otes in existence. They lack a rigid cell wall and generally infect the cytoplasm
of mammalian cells. There are at least five species known to contaminate cells
in culture:Mycoplasma hyorhinis, Mycoplasma arginini, Mycoplasma orale, Myco-
plasma fermentansandAcholeplasma laidlawii.Infections caused by these organisms
are more problematic and not easily identified or eliminated. Moreover, if left
unchecked, mycoplasma contamination will cause subtle but adverse effects on
cultures, including changes in metabolism, DNA, RNA and protein synthesis, morpho-
logy and growth. This can lead to non-reproducible, unreliable experimental results
and unsafe biological products.45 2.4 Aseptic techniques and good cell culture practice