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Preparation of microbiological culture media (preparing nutrient agar slants)

A. The survival and growth of microorganisms depend on available and a favorable

growth environment. Culture media are the nutrient solutions used in laboratories to
grow microorganisms. For the successful culture of a given microorganism it is
necessary to understand its nutritional requirements and then supply it with its
essential nutrients in the proper form and proportions in a culture medium. The
general composition of a medium is as follows:

1. H-donors and acceptors (approximately 1-15 g/L)

2. C-source (approximately 1-20 g/L)
3. N-source (approximately 0,2-2 g/L)
4. Inorganic nutrients e.g. S, P, (50mg/L)
5. Trace elements (0,1-1 µg/L)
6. Growth factors (aminoacids, purines, pyrimidines, occasionally 50 mg/L, vitamins
occasionally 0,1-1 mg/L)
7. Solidifying agent (e.g agar 10-20 g/L)
8. Solvent (usually distilled water)
9. Buffers
According to the consistency three types of media are used: liquid, or broth, media;
semisolid media; and solid media. The major difference among them is that solid and
semisolid media contain a solidifying or gelling agent [such as agar, gelatine], whereas
a liquid medium does not.
− Liquid media, such as nutrient broth, tryptic soy broth or glucose broth can be used
in studies of growth and metabolism in which it is necessary to have homogenous
media conditions, to follow optical density, and to allow early sampling for analysis of
substrates and metabolic products. Tubes and flasks with liquid cultures can be
incubated with either static or shaken incubation.
− Semisolid media can also be used in fermentation studies, in determining bacterial
motility, and in promoting anaerobic growth.
− Solid media, such as nutrient agar, are used
1) for the surface growth of microorganisms in order to observe colony morphology,
2) for pure culture isolation,
3) often in the enumeration and isolation of bacteria from a mixed population by
diluting the original bacteria suspension and spreading a small inoculum over the
surface of the solidified medium and
4) to observe specific biochemical reactions (extracellular enzymes diffusing away
from the colony can be detected as a result of their action on insoluble substrates
present in the agar medium).

Solid media can be poured into either a test tube or Petri dish. If the medium in the test
tube is allowed to harden in a slanted position, the tube is designated an agar slant; if
the tube is allowed to harden in an upright position, the tube is designated an agar deep
tube; and if the agar is poured into a Petri dish, the plate is designated an agar plate.
Media are categorized by their composition:
− Chemically defined, or synthetic, media are composed of known quantity and
quality of pure chemicals.
− In routine bacteriology laboratory exercises, complex or nonsynthetic media are
employed. These are composed of complex materials rich in vitamins and nutrients,
the chemical composition of which is poorly defined. Three of the most commonly
used components are beef extract, yeast extract and peptone (partially digested
protein). Media are categorized by their function:
− An all-purpose medium, such as Tryptic Soy Agar, supports the growth of most
bacteria cultured in the laboratory. They do not contain any special additives.
− Selective media enhance the growth of certain organisms while inhibiting the
growth of others due to the inclusion of particular substrate.
− Differential media allow identification of microorganisms usually through the
(visible) physiological reactions unique to those bacteria. The most practical media are
those that both select for and differentiate common pathogens.
− Enrichment media allow metabolically fastidious microorganisms to grow because
of the addition of specific growth factors. Enrichment culture is one obtained with the
use of selected media and incubation conditions to isolate the desired microorganisms
from natural samples.
The preparation of media from commercially available dehydrated products is simple
and straightforward. Each bottle of dehydrated medium has instructions for
preparation of its label. For example, to prepare a liter of tryptic soy broth, suspend 30
g of the dehydrated medium in 1.000 ml distilled water. Mix thoroughly in a 2 liter
Erlenmeyer flask [always use a flask that holds twice the volume of media you are
preparing]. Dispense and sterilize for 20 minutes at 121 0 C [15 lbs pressure]. As
noted, the amount of powder for 1.000 ml of water will be indicated. In case of
preparation of media from a formula pons 500 ml of distilled water into a 2000ml
Erlenmeyer flask. Then measure adequate amount of media components and dissolve
completely in the water in the order of the formula. At the end rins the flask with the
remaining 500 ml water. Mix the medium thoroughly, adjust the pH and sterilize.

If the medium lacks agar, the powder will usually dissolve without heating. If it
contains agar, it is necessary to heat the medium until it starts to boil or even longer in
order to completely dissolve the agar. Most of the exercises you will be doing in this
manual will involve the use of sterile media culture tubes. These tubes must be capped
in order to maintain media sterility. This can be accomplished by using cotton plugs,
plastic foam plugs or plastic or metal caps. All of these caps keep cultures free of
contamination while allowing air into the culture tube, and minimizing evaporation at
the same time. It is sometimes desirable to use screw cap culture tubes. This is
especially true when the culture, such as in the case of slants, may be sealed and stored
for long periods. Culture broth can be dispensed with the pipetting machine, an
automatic syringe, or a regular pipette. Agar deep tubes can be stored after sterilization
for use in the preparation of Petri plates when needed. Some agar deeps may be stored
at room temperature for several days before use. If longer periods of storage are
required, they should be placed in the refrigerator in order to prevent drying of the
agar. When Petri plates are needed, the agar deeps are melted either in a boiling water
bath or by bringing them to 121 0 C in an autoclave for 30 to 60 seconds, and then
releasing the steam under slow exhaust. After the agar has melted, the pours are
transferred to a 48 to 50 0 C water bath and kept there for at least 5 to 10 minutes
before use.

The agar deeps should be cooled to about 50 0 C before they are used to minimize
the amount of steam condensation on the Petri plate lids after the agar has been
poured. Agar does not solidify until its temperature drops to about 42 0 C. When the
deeps have reached 50°C , one is taken from the bath and the outside is dried with a
paper towel. Its cap is removed and the top 9 is briefly flamed using a Bunsen burner.
The agar is immediately poured into a sterile Petri plate while holding the top
carefully above the Petri plate bottom in order to avoid contamination. Replace the
top, allow the agar to cool and harden, and store the Petri plates in an inverted