Lab Module 7: Culture Media
INTRODUCTION
Many different types of media are available for growing bacteria. You are familiar with two allpurpose 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 or halt 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. There are many kinds of selective media.
Differential media contain chemicals that produce visible differences among different groups of
organisms when grown on that medium. These differences are unrelated to how well the
organisms grow on the medium. 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 Plates.
Most common bacteria can survive in environments with solute concentrations up to a few
percent (2-3%). Agar containing 7% NaCl presents an osmotic pressure that is too extreme for
many bacteria to survive on. Organisms in the genus Staphylococcus, however, have [poorly
understood] mechanisms for withstanding these higher osmotic pressures. Thus, 7% NaCl Agar is
a selective medium.
Procedure:
1. Label and streak for isolation a 7% NaCl Agar plate with Staphylococcus epidermidis.
2. Label and streak for isolation a 7% NaCl Agar plate with Enterobacter cloacae.
3. Incubate the two plates at 30C.
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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 for by using agar plates containing sheep’s blood
(or the blood of some other animal): hemolysis is seen as a change of the agar surrounding the
colonies. There are three identifiable levels of hemolytic ability:
1. β (beta) hemolysis = complete hemolysis. The agar is “cleared” around the colonies. The
cleared agar will have a yellow tint to it, but you will be able to see through it.
2. α (alpha) hemolysis = partial hemolysis. The agar surrounding the colonies normally
darkens in color compared to areas of the plate where colonies are not present, but the
agar will not be see-through. Sometimes, you may also see a greenish tint in the agar
around the colonies.
3. Negative (– also called gamma, γ) hemolysis = No hemolysis has occurred, although there
should be visible colonies. The agar should still be a bright blood red color.
Procedure:
1. Label and streak for isolation a BAP with Staphylococcus aureus.
a. NOTE: this bacterium may instead only be demonstrated by the instructor
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 color when pH changes. This is useful
because acids are almost always produced (pH decreases) when fermentation occurs. Organisms
capable of fermenting lactose (the carbohydrate present in EMB plates) form dark or hot pink
colonies, while lactose non-fermenters form clear or light colored 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 ability) – they are
like running two separate tests at the same time.
Procedure:
1. Label and streak for isolation an EMB plate with Staphylococcus epidermidis.
2. Label and streak for isolation an EMB plate with Pseudomonas fluorescens.
3. Label and streak for isolation an EMB plate with Enterobacter cloacae.
a. NOTE: the instructor may also demonstrate with Escherichia coli.
4. Incubate the three plates at 30C.
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DAY TWO ACTIVITIES
Activity 1: Observation of 7% NaCl Plates.
1. Observe the S. epidermidis and E. cloacae plates.
a. Which organism is salt-tolerant?
Activity 2: Observation of blood agar plates.
Note: The pictures below should help you determine the hemolytic ability of each organism.
1. Observe the S. epidermidis plate by holding the plate up toward a light (like the ceiling
lights) and looking through the back of the plate.
a. Is S. epidermidis alpha, beta, or negative for hemolysis?
2. Observe the S. aureus plate in the same manner.
** NOTE: do NOT open the S. aureus plate. Observe it with the lid closed.**
a. Is S. aureus alpha, beta, or negative for hemolysis?
β hemolysis
α hemolysis
Activity 3: Observations of EMB plates.
α, β, and γ (-) hemolysis
1. Observe the three EMB plates. Describe the growth on the three plates.
a. Plate with S. epidermidis:
b. Plate with E. cloacae:
c. Plate with P. fluorescens:
2. Which species is/are Gram-positive?
3. For the Gram-negative species, which is/are positive for lactose fermentation?
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QUESTIONS
1. Of the two selective media used in this module, which is more strictly selective?
2. For the more moderately selective plate, how do you determine the Gram reaction of a
particular organism?
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.
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