Once the microscopic morphology and staining characteristics of a microorganism present in a
clinical specimen are known, the microbiologist can make appropriate decisions as to how it should
be cultivated and what biological properties must be demonstrated to identify it fully.
First, a suitable culture medium must be provided, and it must contain the nutrients essential
for the growth of the microorganism to be studied. Most media designed
for the initial growth and isolation of microorganisms are rich in protein components derived from
animal meats. Many bacteria are unable to break down proteins to usable forms and must be provided
with extracted or partially degraded protein materials (peptides, proteoses, peptones, amino
acids). Meat extracts, or partially cooked meats, are the basic nutrients of many culture media.
Some carbohydrate and mineral salts are usually added as well. Such basal media may then be supplemented,
, with blood, serum, vitamins, other carbohydrates and mineral salts, or particular
amino acids as needed or indicated.
In this exercise, we will prepare a basic nutrient broth medium and also a nutrient agar
from commercially available dehydrated stock mixtures containing all necessary ingredients except
water. The term nutrient broth
) refers specifically to basal media prepared from meat extracts
with a few other basic ingredients, but lacking special enrichment. We will also see how liquid and
agar media are appropriately dispensed in flasks, bottles, or tubes for sterilization before use, and
how a sterile agar medium is then poured aseptically into petri dishes.
||To learn how culture media are prepared for use in the microbiology laboratory
||Dehydrated nutrient agar
Dehydrated nutrient broth
A balance, and weighing papers
A 1-liter Erlenmeyer flask, cotton plugged or screw capped
A 1-liter glass beaker
A 1-liter graduated cylinder
Glass stirring rods (at least 10 cm long)
10-ml pipettes (cotton plugged)
Test tubes (screw capped or cotton plugged)
Aspiration device for pipetting
- Read the label on a bottle of dehydrated nutrient agar. It specifies the amount of dehydrated powder required to make
1 liter (1,000 ml) of medium. Calculate the amount needed for 1/2 liter and weigh out this quantity.
- Place 500 ml of distilled water in an Erlenmeyer flask. Add the weighed, dehydrated agar while stirring with a glass rod to
- Set the flask on a tripod over an asbestos mat. Using a Bunsen flame, slowly bring the rehydrated agar to a boil. Stir often.
An electric hot plate may be used instead of a Bunsen burner.
- When the agar mixture is completely dissolved, remove the flask from the flame or hot plate, close it with the cotton plug
or cap, and give it to the instructor to be sterilized in the autoclave.
- While the flask of agar is being sterilized, prepare 500 ml of nutrient broth, adding the weighed dehydrated powder to the
water in a beaker for reconstitution and dissolution.
- Bring the reconstituted broth to a boil, slowly. When fully dissolved, remove from flame or electric burner and allow to
cool a bit.
- The instructor will demonstrate the use of the pipetting device. Do not pipette by mouth. Using a pipette, dispense 5-ml
aliquots of the broth into test tubes (plugged or capped). The instructor will collect the tubes and sterilize them.
- When the flask of sterilized agar is returned to you, allow it to cool to about 50°C (the agar should be warm and melted,
but not too hot to handle in its flask). Remove the plug or cap with the little finger of your right hand and continue to
hold it until you are sure it won’t have to be returned to the flask. Quickly pour the melted, sterile agar into a series of
petri dishes. The petri dish tops are lifted with the left hand, and the bottoms are filled to about one-third capacity with
melted agar (fig. 8.1). Replace each petri dish top as the plate is poured. When the plates are cool (agar solidified), invert
them to prevent condensing moisture from accumulating on the agar surfaces.
- Place inverted agar plates and tubes of sterilized nutrient broth (cooled after their return to you) in the 35°C incubator.
They should be incubated for at least 24 hours to ensure they are sterile (free of contaminating bacteria).
|Figure 8.1 Preparing a plate of agar medium by pouring melted sterile agar into it.