Sterilisation – Preparation of Culture Media
Nutrient media – the basis for reproducible results
Microorganisms can only be determined precisely subsequent to being cultured in a pure culture medium. The
precondition for this is optimal nutrient media. Correct
selection of the nutrient medium but also its quality are
determined to a large extent by the quality of the culture.
The quality of a particular medium is largely determined
by its batch constancy which is the basis for reproducible
results.
Composition of nutrient media
The most important components of the nutrient medium
are carbohydrates as a source of carbon, peptones as a
source of nitrogen, minerals as enzyme activators and
for maintaining osmotic pressure and growth factors
(essential amino acids, vitamins etc.). Solid media are
prepared by adding 1.5 to 2 % of agar-agar, an extract
from algae of the species gelidium, or 10 to 15 % gelatine.
As each microorganism has its own optimum pH range,
buffer systems, mostly phosphate buffers, are normally
added. This has the effect of buffering acids resulting
from the degradation of carbohydrates.
Types of nutrient medium
Nutrient media can be differentiated according to the
components involved, their consistency and their function.
Components
One can differentiate between media containing natural
biological components and those that are categorised as
either semi- or fully synthetic. As biological materials are
always subject to a certain degree of fluctuation and
which are undesired in the case of certain determinations (e.g. sensitivity testing), media have been developed consisting either partially or wholly of precisely
defined chemical substances.
Consistency
One can differentiate between solid media usually containing 1.5 to 2 % agar, semi-solid media containing less
than 1 % (e.g. for mobility testing) and nutrient broths.
Function
● Universal or basic media
These are complex or synthetic non-selective media
used for the isolation of numerous different
microorgnisms.
● Elective media
These are media which promote the growth of certain
microorganisms.
● Selective media
These inhibit the growth of certain microorganisms.
● Differentiating media
Media which enable various microorganisms to be
differentiated on the basis of pH or redox indicators.
● Enhancement media
Liquid media designed to achieve high organism
densities, both as selective and non- selective media.
● Strain-maintenance media
For the cultivation of reference media.
Manufacture of nutrient media
Dissolving
Almost all known nutrient media are available in a premixed and tested form and supplied by commercial companies. Ready-to-use solutions, concentrates or preprepared mixtures of powders are available. Dry media are
“dehydrated“ products and may be strongly hygroscopic. They are dissolved in demineralised or distilled water
at a pH of 7. Dehydrated products are those from which
water has been removed during the manufacturing
process. Dry nutrient media must be closed in their original packages and stored air-tight.
In preparing such media, the instructions provided by the
manufacturer should be followed precisely. The required
quantity of dry substance should be suspended in the
prescribed volume of water and allowed to stand for
approximately 15 minutes so that the agar/gelatine mixture can swell. To complete the solution, the suspension
should be stirred using a magnetic stirrer, warmed and
subsequently sterilised. This is normally done by autoclaving for 15 minutes at 121 °C. Heat-sensitive media
should be sterilised using steam.
Ready-to-use nutrient media are suitable for use in small
laboratories; they are available commercially and can
be called up from the supplier at regular intervals as and
when required. The expiry date should be kept in mind
when storing.
Automatic preparation of nutrient media
Only a uniform process for preparing nutrient media
can lead to optimal and reproducible results. This is
especially true if larger quantities of media have to be
prepared. Instruments available today enable the dissolving process to be carried out gently, fully automatically controlled and, especially, at the precise tempera-
According to the type of instruments used, 10 l of agarcontaining nutrient medium can be prepared within 60
to 75 minutes and sterilised using the same instrument.
First, water is filled into the stainless steel container and
the required amount of dehydated medium is added. The
sterilisation temperature and time required can be read
off the product package insert.
Subsequent to dissolving and sterilisation, cooling takes
place until the withdrawal temperature is reached and
the medium filled into flasks, bottles etc. In the case of the
preparation of chocolate agar, the entire process can be
automatically controlled. In this case, the temperature of
the medium is reduced subsequent to the first sterilisation
step, blood added at 50 °C and the mixture once more
heated for a short period under stirring at increased temperature (ca. 75 °C). All steps involved in such a process
should be carefully documented. In this way, quality control is guaranteed and possible errors are easier to find.
Especially in the preparation of dry media, control of the
final pH is important. The pH should be adjusted subsequent to sterilisation by adding sterilised 1 N HCL or
1 N NaOH.
The pH should be measured subsequent to the sterilisation process. As a rule, pH indication is based on a
medium temperature of 25 °C. It is recommended that a
precisely measured quantity of sterile product be removed (e.g. 50 ml), as only in this way can correction
with a defined quantity of sterilised 1 N HCL or 1 N
NaOH (or 1/10 N) be carried out.
Media thus prepared should be packed and stored in
plastic bags in order to prevent drying out. A storage temperature of 8-12 °C is recommended as at this temperature water of condensation is not as strong as at 4 °C.
Filling of nutrient media
Nutrient media are normally filled into tubes or dishes
prior to use. In general, tubes should be allowed to cool
in a sloping position so that a larger surface of the
medium becomes available (= sloping agar). Filling can
then take place either manually using a dispenser or by
means of a peristaltic pump.
Manual filling
Manual filling of petri dishes should be carried out in
a seperate and meticulously clean room, if possible in
a laminar flow safety cabinet. Petri dishes of diameter
60 to 100 mm should be filled with 20 to 25 ml of the
liquid medium at a temperature of approximately 45 to
55 °C. At higher temperatures, the dish lids tend to become misted with undesired condensation. Any air bubbles that may appear can be removed by flaming briefly
with a bunsen burner. However, care has to be taken
that substances on the surface of the nutrient media are
not destroyed. The dishes should then be allowed to cool
and, having become solid at 45 °C, should be stored in
a reversed position i.e. with the bottom facing upwards.
This prevents the formation of water of condensation and
hence the removal of water from the medium. Moist agar
surfaces can be dried at approximately 30 to 37 °C
prior to use by placing the bottom of the petri dish with
the inside facing downwards in a sloping position on the
lid.
Automatic filling
If larger amounts of dishes are required, this is best done
using a fully automatic filling apparatus as even experienced personnel may make errors when using manual processes. Using such automatic instruments, up to
1200 petri dishes per hour can be filled with constant
volumes of medium and subsequently stacked. Facilities
equipped with such instrumentation fulfil all requirements
and guarantee constant, uniform and reproducible
quality.
Storage
Prepared media can often only be used within a few
days or even a few hours. Thus, prior to preparing the
medium, one should consider what sort of quantities will
be required. In storing liquid and solid media, loss of
water through evaporation and contamination via
foreign organisms should be avoided at all costs. Prepared media should be stored at around +8 °C in the
dark. Prior to use, the medium should be brought to the
required temperature; any water of condensation present must then be removed prior to inoculation. If the
medium has to be stored for a longer period of time, the
petri dishes should be sealed with adhesive tape and
kept in plastic bags. Tubes should be closed off tightly or
their caps sealed with adhesive tape.
Sterilisation – Preparation of Culture Media
ture (regulated to ± 0.5 °C) required for the quality of the
medium. “Caramelisation“, which brings about a reduction in quality, is hence excluded. Heat-sensitive media
such as blood or chocolate agar can be prepared under
these conditions in uniform quality.