As we have seen, many clinical specimens contain a mixed flora of microorganisms. When these
specimens are set up for culture, if only one isolation plate were inoculated, a great deal of time
would be spent in subculturing and sorting through the bacterial species that grow out. Instead,
the microbiologist uses several types of primary media at once (i.e., a battery) to culture the specimen
initially. In general, the primary battery has three basic purposes: (1) to culture all bacterial
species present and see which, if any, predominates; (2) to differentiate species by certain characteristic
responses to ingredients of the culture medium; and (3) to selectively encourage growth of
those species of interest while suppressing the normal flora.
The basic medium on which a majority of bacteria present in a clinical specimen will
grow contains agar enriched with blood and other nutrients required by pathogens. The blood,
which provides excellent enrichment, is obtained from animal sources, most often from sheep. The
use of human blood (usually obtained from outdated collections in blood banks) in culture media
is not recommended because it may contain substances such as antimicrobial agents, antibodies,
and anticoagulants that are either inhibitory to the growth of fastidious microorganisms or interfere
with characteristic reactions.
In addition to basic nutrients, differential media
contain one or more components, such
as a particular carbohydrate, that can be used by some microorganisms but not by others. If the microorganism
uses the component during the incubation period, a change occurs in an indicator
that is also included in the medium (see colorplate 16 and 17
contain one or more components that suppress the growth of some microorganisms
without seriously affecting the ability of others to grow. Such media may also contain
ingredients for differentiating among the species that do survive.
When a battery of several culture media such as just described is streaked upon receipt
of a clinical specimen, the first results indicate what types of bacteria are present, in general how
many, and which did or did not use the differential carbohydrate. Also, the species of particular interest
on the selective medium (if that species was present in the specimen) has been singled out
and differentiated. Thus, the process of identification of isolated pathogens is already well under
way after 24 hours of incubation of specimen cultures.
||To observe the response of a mixed bacterial flora in a clinical specimen to a battery of primary
||Nutrient agar plates
Blood agar plates
Eosin methylene blue agar plates (EMB)
Mannitol salt agar plates (MSA)
Simulated fecal suspension, containing Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus epidermidis
Mannitol salt plate streaked with Staphylococcus aureus on one side (pure culture), Escherichia coli on the other (pure culture)
Eosin methylene blue plate streaked with Staphylococcus aureus and Escherichia coli
Table 16.1 summarizes the most commonly used enriched, selective, and differential
media, indicating their purpose as primary media for the isolation of microorganisms. The table
should be reviewed before performing the exercise.
|Table 16.1 Culture Media for the Isolation of Pathogenic Bacteria from Clinical Specimens
- Inoculate the simulated fecal specimen on nutrient agar, blood agar, EMB, and MSA plates. Streak each plate for isolation
of colonies. Incubate at 35°C.
- Make a Gram stain of the fecal suspension and examine it.
- Examine the demonstration plates (do not open them) and record your observations.
- Demonstration plates:
- Describe the appearance of S. aureus on
- Mannitol salt agar
- EMB agar
- Describe the appearance of E. coli on
- Mannitol salt agar
- EMB agar
- Simulated fecal specimen cultures.