Choosing a Microbiology Laboratory Autoclave/Sterilizer
- Autoclaves or sterilizers are vessels that use pressurized steam to eradicate micro-organisms.
- Their purpose in the microbiology laboratory is either to prepare culture media, reagents and equipment
- or for decontaminating biohazardous waste materials.
Whilst sterilization can be achieved using
a number of techniques: irradiation [UV and other]; filtration; chemical etc.
In the microbiology laboratory, the most widely used sterilizers are autoclaves
- these eradicate micro-organisms by use of pressurized steam to cause thermal
stress. To achieve a sufficient kill rate it is necessary to raise the
temperature such that even the most thermo-tolerant are inactivated, hence the
classic 121°C for 15 minutes.
With the increased requirement to meet various laboratory safety and quality
standards, modern autoclaves have evolved considerably from the days when large
'pressure cookers' were widely used, this has resulted in a whole range of
autoclave alternatives, choosing one will depend on your finance as well as the
primary purpose for which the autoclave will be used.
There is a need to verifiably demonstrate that not only have organisms been
eradicated but also, in particular with culture media, that the growth
chracteristics of the media have not been compromised by the sterilization
cycle. It can be virtually guaranteed that some components will be thermolabile
and suffer from a degree of heat degradation during the
autoclaving/sterilization process, possibly leading to reduced performance.
Consequently there are 2 main considerations when sterilizing culture media:
- Is the thermal cycle sufficient to eradicate micro-organisms?;
- Is the culture medium still usable?
These 2 functions can frequently conflict. However commercially available
culture media usually come with recommended sterilization procedures, which if
followed will allow optimal sterilization and ensure that the culture media
performance is not compromised. Accelerated cooling systems means the time at
high temperatures is kept to a minimum and for added convenience some
autoclaves will have a warming function for plating media that can maintain
agar at 40°C ready for pouring when required.
Waste disposal is less demanding in that a definitive 'overkill' policy can be
used i.e. temperature/time/pressure such that no organisms can survive the
thermal cycle. But there must be adequate steam penetration of the load which
can be difficult to achieve when there are large volumes of air such as with
petri dishes or porous loads.
It is important to think about
what you are going to put into the autoclave to make sure that the one you buy
has the right specification to process it effectively and efficiently,
especially if you are going to have to prove this to a certifying body later
The most critical element is the loading. Placing too much material in an
autoclave does not allow sufficent room for thorough steam circulation - how
can you be certain that all the load has been adequately sterilized? Make sure
your system purchase will cope with the volume and type of material you need to
A pre-cycle vacuum removes air from the load, free-steaming provides better
temperature distribution and pulsed free-steaming improves steam penetration.
For porous loads, vacuum drying can be used.Vacuum cooling makes the whole
cycle time faster so more loads can be processed in the working day.
Autoclaves can range in size from small benchtop models which have a small
footprint and therefore take up little laboratory space through to large -
almost industrial scale systems - that would most usually be located in their
own dedicated facility.
The classic design of an
autoclave uses a circular cross section. This enables high pressures to be
achieved with the minimum of stress bearing materials - however the
disadvantage is that more care must be taken in how the autoclave is loaded
particularly with front loading systems. The tallest containers must always be
placed in the centre of the shelf.
Some manufacturers have overcome this by using square or rectangular pressure
vessels. Autoclaves of this type of design can accommodate a variety of loads
more easily but require a considerably more robust construction to withstand
the internal pressure.
With the increasing need for demonstrable validation of laboratory processes,
autoclaves are now available which can semi or fully automate the complete
sterilization cycle and at the same time fully log the characteristics of each
cycle. Microprocessor control systems can be difficult to use so it is
important to consider who is likely to be using the equipment and how often the
settings may need to be changed. Some systems are easy, whereas others require
a manual and passwords to make even a small adjustment to the set time or
Not every laboratory around the world is able to take advantage of the latest
autoclave technology so there remains room for entry level autoclaves - these
are certainly the least expensive but require considerably more labour input.
Inevitably compromises must be made. The more highly specified systems will
cost more money.
How much space do you have?
Lack of floor space would indicate that a benchtop autoclave is more
appropriate - however these will not be able to take as large a load as a free
Then of course there is a choice of front loading or a top loading autoclave?
The latter will not take up as much space but it might be a problem lifting in
or out large flasks of hot media. Conversely a front loading autoclave will
need a little more space - the door has to open outwards.
Talking of doors there are autoclaves with push button door closures - forget
the days of winding up and down butterfly nuts.
Pass Through Rectangular
In some instances there is a
requirement for pass-through autoclaves - these enable loads to be entered from
one side of the autoclave and taken out from the other side - useful for those
environments in which different levels of containment are required, such as
clean rooms or category 3.
For media preparation there are systems which allow multi-cycle run modes -
useful for media such as chocolate agar?
Will you ever need to fully automate the media preparation/plate pouring
process? - if that's likely then it might be appropriate to buy an autoclave
that can integrate at a later date with a plate pourer/bottle filler system.
Autoclaves should be easy to clean both from the inside and outside, some
ranges have coatings which could almost be called 'self-cleaning' - these
incorporate materials which have biocidal activity.
Calibration and Performance Qualification Testing of Autoclaves
for these items:
To succesfully validate an
autoclave there must be certainty that it is functioning as the manufacturer
intended. This can only be achieved by ensuring that it is calibrated and
Calibration is the process of determining the actual* temperature inside an
autoclave when a given temperature is set. Without a calibration - even with
its uncertainties, the actual temperature inside the autoclave is unknown. The
electronics may be wrong, the temperature probe may be damaged, the pressure
gauge - a very useful backup - may also be wrong etc.
Performance qualification is one of several generic terms for testing an
autoclave - and its load - to ensure the load is properly processed. Usually a
calibrated, multi channel, data logger is employed so that considerable numbers
of points in the load, together with salient points on the autoclave, can be
logged. Analysis of these results can point to many problems in the setup of
the autoclave or in the way the load is contained. Significant differences can
be seen, for instance, between glass and plastic bottles or plastic and metal
discard containers, or between single and double bagged discard loads.
Performance qualification is the only way to know what is actually happening to
the load. It is vital that the test load is typical of loads actually processed
as the results cannot necessarily be read across. However, once the performance
qualification is done, calibration alone thereafter is often sufficient - at
least if the load does not change.
Aside from data logging of times and temperatures, there are a number of
techniques using biological and chemical indicators, for ensuring that the
actual load is being adequately sterilized: such as coloured tape, strips,
spore strips and spore suspensions .
Whatever technique is used it must be appropriate to typical loading of the
autoclave both in regard to the load quantity and load contents.
* Where 'actual' means 'best estimate'