Selective and differential Media

Introduction
In addition to general-purpose media, which allow the growth of most types of bacteria,

microbiologists use specialized media to identify and/or isolate specific groups of bacteria. In

this experiment, we will use differential and selective media

Mannitol salt agar (MSA) :

contains 7.5% NaCl, a salt level which is toxic to most bacteria except the genus Staphylococcus. This is
the selective portion of the medium. MSA is also differential. It contains the carbohydrate, mannitol, as
well as the pH indicator, phenol red. Staphylococci that ferment mannitol and produce acid exhibit a
yellow color around their colonies. Staphylococci that do not ferment mannitol do not produce a color
change from the normal red-pink color of the medium.
MacConkey agar (Mac) is used for the isolation of Gram negative organisms. It contains crystal violet,
which inhibits the growth of Gram positive organisms and also gives the medium a light pink-lavender
color. The medium also contains bile salts, lactose, and the pH indicator, neutral red. As well as being
selective for Gram negative, Mac is differential within that group. Coliform bacilli produce acid from
lactose fermentation causing the colonies to turn red from the pH indicator. E. coli producs even greater
quantities of acid causing the surrounding medium and the colonies to turn red. Non-coliform bacilli do
not ferment lactose, and appear uncolored or transparent on Mac.

Levine's eosin-methylene blue agar (EMB) is primarily a differential medium. However, it does inhibit
the growth of some Gram positive bacteria. EMB is used to differentiate between enteric lactose
fermenters (coliforms) and non-lactose fermenters as well as specifically identifying E. coli. It is often
used to confirm the results of tests with MacConkey agar. The eosin and methylene blue dyes cause
lactose fermenters to have pink colonies. E. coli incorporates so much of the dye that the dyes
precipitate in the cells and give the colonies a metallic green sheen. Non-lactose fermenters are usually
transparent and take on the purple color of the media

Materials
Each student/team:

1 Mannitol salt agar plate.

1 MacConkey agar plate.

1 Eosin-methylene blue plate.

Lab supplies:
Nutrient broth cultures of Salmonella typhimurium, Escherichia coli, Enterococcus

(Streptococcus) faecalis and Staphylococcus aureus (all 24- to 48-hour).

Procedure
1. Using a grease pencil or marker, divide the bottom of each of the listed plates into four

sections. Label the plates as shown in Figure 10.1.

2. Using a loop and proper sterile technique, inoculate each plate with E. coli,

Staphylococcus aureus, Salmonella typhimurium and Streptococcus sp. using a single

streak as shown in Figure 10.2.

3. Incubate all of the plates at 37°C for 48 hours. Record the results.