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Kibota 1
Lab Module 7: Culture Media
Introduction
Many different types of media are available for growing bacteria. You are familiar with
two all-purpose agar media: Nutrient Agar with Yeast Extract (NAYE) and Tryptic Soy
Agar (TSA). These two media are rich with nutrients that allow the growth of many
types of organisms. Other kinds of media contain chemicals that cause differences in
bacterial growth.
Selective media contain chemicals that inhibit the growth of some organisms. Other
organisms have enzyme systems that allow them to deal with the selective agents. Thus,
selective media are useful for: (1) isolating a particular group of organisms from a mixed
culture and/or (2) helping to characterize a pure culture. You must keep in mind two
things when using selective media. First, a selective medium isn’t necessarily perfectly
selective—if given enough time, even organisms that are inhibited may reproduce enough
to form visible colonies. Second, even those organisms that are supposed to grow may
form smaller colonies or may grow more slowly than they would on an all-purpose
medium.
Differential media contain chemicals that produce visible differences among different
groups of organisms. Visible differences may take the form of differences in colony
color or changes in the appearance of the culture medium itself. There are many kinds of
differential media. They are useful for helping to characterize and identify bacterial
species.
DAY ONE ACTIVITIES (Performed by pairs of students)
Activity 1: 7% NaCl Agar
Most common bacteria can survive in environments with solute concentrations up to a
few percent (2 to 3%). Agar containing 7% NaCl presents an osmotic pressure that is too
extreme for many bacteria. Organisms in the genus Staphylococcus, however, have
[poorly understood] mechanisms for withstanding these higher pressures. Thus, 7%
NaCl Agar is a selective medium.
1. Label and streak for isolation a 7% NaCl Agar plate with Staphylococcus aureus.
2. Label and streak for isolation a 7% NaCl Agar plate with Enterobacter cloacae.
3. Incubate the two plates at 30°C.
Activity 2: Blood Agar Plates (BAP)
Some bacteria can perform the process of breaking down red blood cells (called
hemolysis). Other bacteria cannot. Hemolytic ability can be tested using agar plates
containing sheep’s blood (or the blood of some other animal): hemolysis is seen as a
clearing of the agar (loss of blood red color) surrounding colonies. There are three
identifiable levels of hemolytic ability:
β Complete hemolysis. The cleared agar is see-through.
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Clark College
Kibota 2
α Partial hemolysis. The agar surrounding colonies is lighter in color than
unaffected agar, but is not see-through.
- Negative. No hemolysis.
1. Label and streak for isolation a BAP with Staphylococcus aureus.
2. Label and streak for isolation a BAP with Staphylococcus epidermidis.
3. Incubate the two plates at 30°C.
Activity 3: Eosin Methylene Blue (EMB) Plates
EMB plates contain a dye that is a pH indicator that changes colors when pH changes.
This is useful because acids are almost always produced (pH decreases) when
fermentation occurs. Organisms capable of fermenting lactose (the carbohydrate
available in EMB plates) form dark colonies while lactose non-fermenters form clear or
light colonies. In addition, the dye is bacteriostatic (slows down the growth) against
Gram-positive species. Thus, EMB plates are both selective (against G+ cells) and
differential (on the basis of lactose fermentation).
1.
2.
3.
4.
Label and streak for isolation an EMB plate with Staphylococcus aureus.
Label and streak for isolation an EMB plate with Enterobacter cloacae.
Label and streak for isolation an EMB plate with Salmonella typhimurium.
Incubate the two plates at 30°C.
DAY TWO ACTIVITIES
Activity 1. Observation of 7% NaCl Plates
1. Observe the S. aureus and E. cloacae plates. Which organism is salt-tolerant?
Activity 2. Observation of BAPs
1. Observe the S. aureus plate by holding the plate up toward a light (like the ceiling
lights) and looking through the back of the plate.
2. Observe the S. epidermidis plate as described above.
β-Hemolysis
- (Negative) Hemolysis
Activity 3. Observations of EMB
1. Observe the three EMB plates. Describe the growth on the three plates.
2. Which species is Gram-positive?
3. For the Gram-negative species, which is lactose fermentation positive?
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Clark College
Kibota 3
QUESTIONS
1. Of the two selective media used in this module, which is more strictly selective?
2. For the more moderately selective plate, how can you tell whether a particular species
shows growth or doesn’t show growth?
3. Describe a natural circumstance where Staphylococcus might benefit from salt
tolerance.
4. How might hemolysis benefit a hemolytic organism?
5. Imagine that you have a culture containing a single bacterial species. This culture
may be one of several possible species. Describe a scenario where you use 7% NaCl,
Blood Agar, and EMB plates to identify the unknown bacterial species.