Selective and Differential Media Lab

Selective Media = Growth Media that contains substances that inhibit certain microorganisms, while allowing other microorganisms
to grow. The presence or absence of growth allows makes it possible to differentiate between the microbes based on their
biochemical characteristics, which determine their sensitivity or resistance to potentially inhibitory substances.
Differential Media = Growth Media that show variations in color following growth of microorganisms, to differentiate between the
microbes based on their biochemical characteristics.

Selective and Differential Agar Plates

Phenylethyl Alcohol Agar (PEA) – This medium makes membranes more permeable, making it difficult for Gram-negative bacteria to
regulate the movement of molecules into and out of the cell, since they have both a plasma membrane and an outer membrane
affected. Thus PEA selects for the growth of Gram-positive bacteria and selects against the growth of Gram-negative bacteria.
MacConkey Agar (MAC) – This medium contains bile salts to inhibit the growth of non-enteric microorganisms. It contains crystal
violet to inhibit the growth of Gram-positive bacteria. Thus MAC selects for the growth of Gram-negative enteric bacteria. The
medium also contains lactose and the pH indicator Neutral Red. Organisms capable of breaking down lactose will create acidic by-
products, causing their growth to turn pink, rather than just taking on the color of the medium (as lactose non-fermenters).
Organisms which produce abundant acid will cause the precipitation of the bile salts in the medium, making their colonies an opaque
pink color.
Mannitol Salt Agar (MSA) – This medium contains a high salt concentration, which inhibits the growth of many bacteria other than
the Staphylocci. The medium contains mannitol as a carbon source. Organisms which ferment mannitol and produce acidic end
products will turn the Phenol Red pH indicator in this medium from red to yellow.
Eosin Methylene Blue Agar (EMB) – This medium contains the dyes Eosin Y and Methylene Blue which partially inhibit Gram-positive
bacteria, while allowing more abundant growth of Gram-negative enteric bacteria. Microbes capable of fermenting lactose will take
on a blue-black color from the dyes, while lactose non-fermenters will remain colorless and transparent. Escherichia coli can be
further distinguished from other enteric bacilli due to excessive production of acids from the breakdown of lactose, resulting in the
precipitation of the dyes from the medium, making the colonies take on a metallic-green sheen.
Procedure for Selective and Differential Agar Plates
1) Label each plate to indicate the type of medium: PEA, MAC, MSA, EMB
2) Use a Sharpie to mark the bottom of each plate, to create three equal zones.
3) Write the names of each of your 3 assigned microbes in the separate zones of the plate.
4) Inoculate each plate using aseptic techniques:
a. Begin with plate upside-down
b. Flame loop
c. Open culture tube, flame lip of tube
d. Collect sample with loop
e. Flame lip of tube, close sample
f. Lift the bottom of the plate and hold open at 45° angle
g. Use the loop to create a line of inoculation in the appropriately labeled zone.
h. Close plate
i. Repeat step g-h for each organism
Multi-Test Media
Triple Sugar Iron Agar Slant (TSIA) – This is a multi-test medium capable of providing several biochemical determinations within one
tube. The three sugars Glucose, Sucrose, and Lactose are each included. The medium also contains iron sulfate salts and the pH
indicator Phenol Red. Organisms capable of metabolizing glucose alone will cause partial yellowing of the medium. Microbes
capable of metabolizing more than one of the sugars will cause both the slant and the butt of the agar to turn yellow. Microbes
which produce Hydrogen sulfide will develop a black color. Microbes capable of producing gas will produce cracks within the
medium.
Sulfide Indole Motility Agar Deep (SIM) – The name of this medium indicates the multi-tests it determines. The production of
Hydrogen sulfide is detected by a black color. Indole production is confirmed by a red ring following the addition of Kovac’s reagent
after incubation. Motility of a microorganism can be detected by growth extending beyond the line of inoculation in the medium.

Procedure for Multi-Test Media

1) Label each tube to indicate the type of medium.
2) Write the names of each of your 2 assigned microbes on the separate tubes.
3) Inoculate TSIA Slants using aseptic techniques:
a. Hold culture tube and TSIA slant with non-dominant hand
b. Flame inoculating needle
c. Open culture tubes, flame lips of tubes
d. Collect sample by dipping needle to the bottom of the culture broth
e. Inoculate TSIA by stabbing center of the medium down to nearly the bottom of the tube
f. Slide the needle from the bottom to the top of the TSIA slant
g. Flame lips of tubes, close tubes
4) Inoculate SIM Deeps using aseptic techniques:
a. Hold culture tube and SIM deep with non-dominant hand
b. Flame inoculating needle
c. Open culture tubes, flame lips of tubes
d. Collect sample by dipping needle to the bottom of the culture broth
e. Inoculate SIM by stabbing center of the medium down to nearly the bottom of the tube
f. Flame lips of tubes, close tubes
Selective and Differential Agar Plates
 Indicate your 3 assigned organisms by circling their names in the table.
 Fill in general information to describe the function of each type of media at the top of the table.
 Inoculate and incubate media with each organism indicated.
 Following incubation, describe growth (or lack of growth), color change, and +/- reaction of interest.

PEA MAC MSA EMB

Type of Media:
Selective,
Differential,
Or Both
Selects For:

Selects Against:

Appearance of
Positive (+)
reaction:

Meaning of Positive
(+) reaction:

Bacillus subtilis

Enterococcus
faecium
Escherichia coli

Pseudomonas
aeruginosa
Staphylococcus
aureus
Staphylococcus
 epidermidis

Multi-Test Media
 Indicate your two assigned organisms by circling their names in the table.
 Indicate Growth Characteristics in Inoculated Agar Deeps:
o Describe appearance/color of growth medium following incubation, or after the addition of reagents.
o Indicate whether each result is a positive (+) or negative (-) reaction.

TSIA SIM

Type of Media:
Selective,
Differential,
Or Both
Growth Medium CO2 H2S H2S Indole* Motility
Color Production Production Production (*add Kovac’s) (+/-)
(+/-) (+/-) (+/-) (color, +/-)
Escherichia coli Slant Color:
Butt Color:
Meaning:

Pseudomonas Slant Color:

aeruginosa Butt Color:
Meaning:

Proteus Slant Color:

mirabilis Butt Color:
Meaning:

Serratia Slant Color:

marcescens Butt Color:
Meaning:
 Clean Up

• Save Lab Manual Data Sheet for next week (following culture incubation)

• Use Sharpie to write your name on tube cylinder. Place all tubes to incubate in tube
cylinder
• Use a single piece of masking tape to hold all plates in a neat stack, bottom/agar sides up.
Add your name and date to the tape, on the top of the stack.
• Incubate all cultures at 37°C – in assigned incubator/shelf

• Used Culture Test Tubes – Remove labels and place in rack next to autoclave
• Used Plates (following data collection) – Large Biohazard bin next to autoclave room

• Disinfect work area with disinfectant in spray bottle, wipe with paper towels.
• Paper Towels – Trash
• Lab Coat, Goggles, Data Sheets – Plastic bag labeled with your name
• Gloves – “Gloves Only” bin beside sink
• Wash hands