PRACTICAL 5

Selective, differential and

enriched media

Date: 11 April 2023

Presenter: Thobeka
Office Number: 108
 Objectives
• To understand the differences between types of media
used in the lab.

• Demonstrate the use of selective and differential media.

• To prepare agar plates.

• To appropriately dispose used agar plates into biological

waste container for incineration.
 4.1 Background:
• Bacteriological media are divided into various categories.

• In terms of physical appearance one can distinguish between solid, semi-solid and liquid
media.
• Nutrient broth is a liquid medium consisting of peptone, yeast extract and NaCl.

• Nutrient agar is a solid medium used for plates in petri dishes and as slants in test tubes. It
consists of a nutrient broth base with 1.5 – 2% agar added as a solidifier.

• The agar contributes little or nothing to the nutritional value of the medium, but it has the
unique property of melting at the boiling point of water (96°C) and only solidifies at about
45°C.

• By reducing the agar concentration to 0.3% one can prepare a semisolid nutrient agar
medium.
 Background cont…
• Nutrient agar is a non-selective, non-enriched, non-differential, general growth medium.
• Various ingredients can be added to ordinary nutrient agar media to give them specific
characteristics.

• The addition of blood, serum, plant and animal tissue extracts, for example, provides
nutrient agar with additional nutrients so that the more fastidious bacteria will grow on it.
• Such media are known as enriched media.

• In selective media certain chemical ingredients are included which permit the growth of
certain kinds of bacteria and inhibit the growth of others.

• Differential media have ingredients which influence the growth of bacteria in a manner
that enables us to distinguish between different species on the same medium.
Examples of selective and/or differential media that are commonly used
include the following:

•Mannitol salts-agar (MS agar): selects for Staphylococcus and differentiates

between S. aureus and S. epidermidis.

•Eosine methylene blue agar (EMB agar): selects for Gram-negative enteric
bacteria and differentiates between lactose fermenters and non-lactose-
fermenters.

•Blood agar: to cultivate fastidious organisms and to differentiate between

organisms that lyse blood cells and those that do not.
 4.1.1 Mannitol salts agar:
• (MS agar) is a selective and differential medium. The selective agent is
sodium chloride at a concentration of 7.5%.
• It inhibits most bacteria, but not those belonging to the genus
Staphylococcus.
• The medium contains 0.5% mannitol and the indicator phenol red.
• When S. aureus grows in this medium, it ferments the mannitol and
releases acid by-products which in turn change the pH indicator in the
surrounding medium from red to yellow.
o S. epidermidis in contrast does not ferment mannitol and will thus not
cause a change in the pH indicator.
• By observing the colour change it is thus possible to differentiate between
S. aureus and S. epidermidis.

Cultures used:
•Bacillus subtilis
•Escherichia coli
•Staphylococcus aureus
•Staphylococcus epidermidis
 4.1.2 Eosine methylene blue(EMB) agar:

• EMB agar is also a selective and differential medium.

• Contains 0.065 g/l methylene blue and 0.4 g/l eosine dyes at concentrations
that will inhibit the growth of most Gram-positive bacteria.
• It differentiates between Gram-negative bacteria that can
ferment lactose (E. coli, Enterobacter and Klebsiella) and those that
cannot ferment lactose (Salmonella, Shigella and Proteus).
• Lactose fermenting organisms form dark purple colonies or pink
colonies with dark centres.
• Species that produces high levels of acid during fermentation (E. coli)
precipitates the dyes and a metallic green sheen forms over the colony.
• Low acid producers can only precipitate a small amount of the dyes and
form pink colonies with dark centres.
 4.1.3 Blood agar:
• Blood agar is a differential medium that supports the growth of a wide variety of bacteria.
• The medium consists of a nutrient rich base with 5% sheep red blood cells.
• Blood agar is commonly used in laboratories to differentiate between organisms that lyse blood
(haemolytic) and organisms that do not
lyse blood (non-haemolytic).
• The breakdown of red blood cells can be observed as a clear zone
around colonies. When the red blood cells are completely lysed and the medium around the
colonies is clear, this type of haemolysis is known as beta-haemolysis. If the red blood cells are
only partially
lysed and the medium has a greenish appearance, this type of haemolysis is known as
alphahaemolysis.
• Non-haemolytic bacteria cause no observable change and this is known as gammahaemolysis.
• Bacteria that are commonly differentiated based on haemolytic reactions are streptococci and
staphylococci.
• Haemolysis occurs as a result of an enzyme secreted by the microorganism.
 4.2.1 Methodology:
Preparation of Nutrient agar –
•Suspend 28g of nutrient agar powder in 1L of distilled water.
•Mix and dissolve completely.
•Sterilize by autoclaving at 121°C for 15 minutes.
•Pour the liquid into the petri dish and wait for the medium to solidify. Be sure that
you are preparing the agar in the clean environment to prevent any contamination.
•Make a simple inoculation line/streak of each culture onto each of the media.
After incubation -
•Compare media before and after incubation
•Pay attention to:
 Growth or no growth of cultures
 Colour change of the media
 Colour change of the cultures
 4.2.2 Methodology:
Preparation of MS agar –
•Suspend 111 grams of Mannitol Salt Agar in 1L of distilled water.
•Boil to dissolve the medium completely.
•Sterilize by autoclaving at pressure (121°C) for 15 minutes.
• If desired, sterile Egg Yolk Emulsion (E7899) can be added to a final concentration
of 5% v/v after autoclaving.
•Make a simple inoculation line/streak of each culture onto each of the media.
After incubation -
•Compare media before and after incubation
•Pay attention to:
 Growth or no growth of cultures
 Colour change of the media
 Colour change of the cultures
 4.2.3 Methodology:
Preparation of EMB agar–
•Suspend 36 grams of EMB Agar in 1000 mls of distilled water. Heat to dissolve the
medium completely.
•Dispense and sterilize by autoclaving at 15 lbs. pressure (121 °C) for 15 minutes.
•Avoid overheating. Cool to 50 °C and shake the medium in order to oxidize the
methylene blue (i.e. to restore its blue colour) and to suspend the flocculent precipitate.
•Make a simple inoculation line/streak of each culture onto each of the media.
After incubation -
•Compare media before and after incubation
•Pay attention to:
 Growth or no growth of cultures
 Colour change of the media
 Colour change of the cultures
 4.2.4 Methodology:
Preparation of Blood agar –
•Suspend 28 g of nutrient agar powder in 1 litre of distilled water.
•Autoclave the dissolved mixture at 121 degrees Celsius for 15 minutes.
•Allow to cool but not solidify.
•When the agar has cooled to 45-50 °C, add 5% (vol/vol) sterile defibrinated blood that has
been warmed to room temperature and mix gently but well.
•Dispense into sterile plates while liquid.
•Make a simple inoculation line/streak of each culture onto each of the media.
After incubation -
•Compare media before and after incubation
•Pay attention to:
 Growth or no growth of cultures
 Colour change of the media
 Colour change of the cultures
 4.3 Results before Inoculation
Nutrient Agar MS Agar

EMB Agar Blood Agar

 4.4 Results After Inoculation- Nutrient
1. Bacillus subtilis
Agar: 2. Escherichia coli

3. Staphylococcus aureus 4. Staphylococcus epidermidis

S. aureus –
can also
produce
Pigment --->
4.5 Results After Inoculation- MS agar:

Staphylococcus
aureus,

Staphylococcus
epidermidis

Can be either
Bacillus subtillis or
Escherichia coli
4.6 Results After Inoculation- EMB agar:

This is how results

will look for:

Staphylococcus aureus

Staphylococcus epidermidis
Bacillus
subtilis

E.coli:
 4.7 Results After Inoculation- Blood agar:
A. Bacillus subtilis B. E.coli

C. Staphylococcus aureus D. Staphylococcus epidermidis

 4.8 References:
• https://youtu.be/9NdYHsJtb0M - Preparation of Nutrient Agar and
pour plate technique
• https://youtu.be/ON4qQafGcfc - Mannitol Salt Agar (MSA) | Results

• https://youtu.be/rWx-lHyEhek - E. coli on EMB agar - colony

morphology results

• https://youtu.be/uVU1JXfXwVk - Hemolysis/Blood Agar