CBLG151

Laboratory 3 Preparation and Use of Culture Media Microbiology I

Laboratory 3

PREPARATION AND USE OF CULTURE MEDIA

Introduction

In order to grow or cultivate microorganisms in the laboratory, it is necessary to provide

the cells with the required nutrients and proper environmental conditions for growth.
Nutrients include the raw materials used to sustain living organisms, replace cellular
components and provide energy for metabolic reactions. All living systems require
nutrients that provide a source of C, N, O, H, P, S, together with essential cations such
as Na+, K+, Mg2+ and Ca2+. A typical culture medium consists of water, a carbon and
energy source (e.g. glucose), and sources of inorganic salts (N, P, S), oxygen, hydrogen
and various metal ions as well as certain growth factors (vitamins, amino acids,
nucleosides). A culture medium may be liquid (broth) or solid (agar).

When a culture medium has been prepared it is not ready for use until it has been
sterilized. Microorganisms associated with the raw ingredients, glassware and in the air
and water are likely to be a source of contamination. If a non-sterile medium is left for a
few days, the contaminating microorganisms will grow in the medium and consume the
nutrients. The process of sterilization is intended to remove or kill all living
microorganisms in the culture medium before it is used.

Culture media can be grouped by its chemical composition, nutritional nature or functional
nature.

A. Chemically defined and complex media:

In a chemically defined medium, the identity and amounts of each component used to
make up the medium is known. In a complex medium, the chemical composition of the
medium is not known. The use of beef or yeast extract produces a complex medium
since the exact contents of the extract are not known-you only know that the extract will
support the growth of microorganisms
.
B. Minimal and nutritionally rich media:

A minimal medium supplies only the essential minimal nutritional requirements to

support the growth of microorganisms and nothing extra. A nutritionally rich medium
contains a variety of nutrients, far above the minimal requirements of a microorganism.
Most microorganisms will grow faster on rich media compared to minimal media because
less time and resources are spent producing basic building blocks of the cell such as
amino acids, nucleosides, fatty acids and vitamins (nutrients usually supplied in a rich
medium).

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Laboratory 3 Preparation and Use of Culture Media Microbiology I

C. Selective, differential and enriched media:

The functional classification of media is used to identify the purpose for which the
medium is being used. Functional media can be selective, differential (or both) or
enriched.

A selective medium supports the growth of a desired microorganism while

inhibiting the growth of others. Selectivity can be obtained by adding a compound that
inhibits undesired microorganism (e.g. crystal violet will inhibit the growth of Gram-
positive bacteria; antibiotics; high levels of salt) or by deleting a compound required by
the undesired microorganism (e.g. glucose; growth factor).

A differential medium supports the growth of many different types of microorganisms

but distinguishes them by their appearance after growth in the medium. For example, the
addition of lactose and a pH indicator (neutral red) to nutrient agar would differentiate
between bacteria growing in the medium and producing acid from lactose metabolism
(red colonies) and bacteria growing in the medium not producing acid (white colonies).

An enrichment medium enhances the growth of a particular microorganism in a mixed

population of microorganisms. For example, the addition of a compound to the medium
allows the desired microorganism to outgrow its competitors. If the compound added to
the medium lowered the pH to 4, then acid tolerant organisms (yeast and molds) would
have a growth advantage over acid-sensitive organisms (most bacteria).

Examples of selective, differential and enriched media that will be used in this lab are
outlined below.

MacConkey Agar: This medium is both selective and differential in nature. The
ingredients which make the medium selective are crystal violet and bile salts (both inhibit
Gram-positive bacteria). MacConkey agar is differential in that this medium distinguishes
between lactose-fermenters (form red colonies e.g. Escherichia coli) and non-lactose-
fermenters (form white/cream colonies e.g. Providencia rettgeri). The presence of lactose
and the pH indicator neutral red (turns red in acid) make the medium differential.

Mannitol Salt Agar: This medium is both selective and differential. The selective
ingredient is NaCl, present at a concentration of 7.5%. NaCl inhibits many bacteria except
those of the genus Staphylococcus. This medium also contains 0.5% mannitol (another
type of carbohydrate-know the structure of glucose and mannitol!) and the pH indicator
phenol red (yellow in acid). When Staphylococcus aureus grows on the medium, the
bacteria ferment the mannitol releasing acids which cause the pH indicator in the agar to
change to yellow. Staphylococcus epidermidis grows on the medium but does not
ferment the mannitol, and therefore, the medium remains red.

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Laboratory 3 Preparation and Use of Culture Media Microbiology I

Nutrient Agar + NaCl: This medium consists of nutrient agar plus NaCl at a
concentration of 7.5%. Like mannitol salt, it is selective for the staphylococci. However,
there is no differential agent in the agar. Although the medium has a high concentration
of nutrients (contained in the nutrient agar) favouring the growth of a wide variety of
bacteria, only organisms which are salt-tolerant (halotolerant) will grow.

Blood Agar: This is an enriched medium used to grow nutritionally demanding

microorganisms, and also differential medium that distinguishes between bacteria that
lyse red blood cells (hemolytic bacteria) and those that do not (non-hemolytic bacteria).

Blood agar is a nutrient-rich medium containing 5% sheep red blood cells. When red
blood cells are lysed completely and the medium around the colony is clear, this type of
hemolysis is called beta-hemolysis. If red blood cells are partially lysed and the medium
acquires a greenish hue around the colony, this is referred to as alpha-hemolysis. Non-
hemolytic bacteria cause no visible change in the medium and this is called gamma-
hemolysis. Hemolysis occurs as a result of the action of specific exo-enzymes released
by bacteria called hemolysins.

Chocolate Agar: This is an enriched medium. It contains heated blood (which turns a
chocolate colour). Heat releases the heme from hemoglobin and inactivates the enzyme
that hydrolyses NAD, two nutrients that some bacteria require for growth.

Learning Objectives:

1. Be able to prepare and sterilize media.

2. Be able to understand the differences and similarities between selective, differential
and enriched media.
3. Be able to identify a given unknown bacteria, by deduction of how the culture
grows on various media.
4. Be able to define or explain the following terms:
Culture media (medium) Cultivate
Chemically defined media Complex media
Minimal media Nutritionally rich media
Selective media Differential media
Enriched media Contamination
Sterilization Autoclave
Membrane filtration Heat-sensitive solutions
Selective agent Bacteriostatic
pH indicator Crystal violet
MacConkey agar Mannitol salt agar
Nutrient agar + NaCl Blood agar
Hemolysis (alpha, beta and gamma)

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Laboratory 3 Preparation and Use of Culture Media Microbiology I

Experiments:

3.1 Preparation of Selective Nutrient Agar

3.2 Selective, Differential and Enriched Media

Bacterial strains used in the lab:

Bacillus subtilis (B. subtilis) Staphylococcus epidermidis (S. epidermidis)
Escherichia coli (E. coli) Enterococcus faecalis (E. faecalis)
Providencia rettgeri (P. rettgeri) Streptococcus lactis (S. lactis)
Staphylococcus aureus (S. aureus) *Haemophilus influenzae (H. influenzae)

*H. influenzae will not be handled by students due to its pathogenecity. You lab instructor
will inoculate this bacteria on your plates under the Biosafety Cabinet.

Experiment 3.1 Preparation of Selective Nutrient Agar

The stain crystal violet can be added to a medium to inhibit the growth of Gram-positive
bacteria. This medium would be selective for Gram-negative bacteria. Crystal violet, like
many stains, is bacteriostatic and at high concentrations it will inhibit all bacterial growth.
It is possible to reduce the concentration of the stain to a level which suppresses the
growth of only the Gram-positive bacteria.

Materials
▪ 250 mL flask
▪ nutrient broth powder
▪ agar powder
▪ hot plate stirrer
▪ aqueous solution of crystal violet (1/25,000)
▪ 2 tubes of 9.0 mL sterile water
▪ 1.0 mL pipettes
▪ 4 sterile plastic petri dishes

Procedure

1. Weigh out 0.8 grams of Nutrient broth powder and place in flask. Add 1.5 grams of
agar powder.
2. Add 100 mL of dH20 and bring the mixture to boiling to dissolve the agar.
3. Autoclave at 121oC for 20 minutes.
4. Meanwhile, mix crystal violet solution thoroughly. Make 1/10 and 1/100 dilution of
the stain solution to give final dilutions of 1/250,000 and 1/2,500,000.

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Laboratory 3 Preparation and Use of Culture Media Microbiology I

5. Label 3 Petri plates and add 1.5 mL of each of the three crystal violet dilutions to the
empty plates. Leave the fourth dish empty.
6. After autoclaving, pour approximately 25 mL of warm agar into each of the petri
dishes. Leave the plates on the bench until hardened.
7. After the plates have hardened, divide the plates in half by drawing a line in the
bottom of the plate. Label and inoculate one half of each plate with E. coli and the
other half with B. subtilis.
8. Incubate the plates at 37oC for 24 hours.

Experiment 3.2 Selective, Differential and Enriched Media

Materials
1 plate each of:
▪ MacConkey agar
▪ Mannitol salt agar
▪ Nutrient Agar + NaCl
▪ Blood agar
▪ Chocolate agar

Procedure

1. Divide the MacConkey, the Mannitol Salt and the Nutrient agar + NaCl plates into 3
sections and label each section with the appropriate name of the organism.

a) Inoculate the MacConkey agar plate with E. coli, Staphylococcus aureus and
P. rettgeri.
b) Inoculate the mannitol salt agar plate with E. coli, Staphylococcus aureus and
Staphylococcus epidermidis.
c) Inoculate the nutrient agar + NaCl plate with E. coli, Staphylococcus aureus
and Bacillus subtilis.

2. Divide the blood agar and the chocolate agar into four sections and label each
section with the appropriate name of the organism.

a) Inoculate both agar plates with Staphylococcus aureus, Enterococcus faecalis,

and Streptococcus lactis. Leave the quadrant uninoculated for Haemophilus
influenza*. The H. influenza* will be inoculated for you under the biosafety
cabinet. Please make sure you have a labeled space on your agar plates
so that it is clear where it will be inoculated into.

3. Incubate these plates at 37oC for 24 hours.

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Laboratory 3 Preparation and Use of Culture Media Microbiology I

NEXT WEEK

Examine all your plates carefully. Record all results.

At the end of Laboratory 3, you should know the following:

1. All the different types of media: chemically defined, enriched, complex, minimal,
selective, differential.
2. The selective agents in: MacConkey Agar, Mannitol Salt Agar and Nutrient Agar +
7.5% NaCl.
3. The differential agents in: MacConkey Agar, Mannitol Salt Agar; Blood Agar
4. Preparation of selective media.
5. Identification of bacteria using different types of media.

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