PRACTICAL NO.

5
ISOLATION OF FOODBORNE PATHOGENS ON SELECTIVE, DIFFERENTIAL, AND
ENRICHED MEDIA
OBJECTIVE
- Learn the properties of, and the uses for, selective and differential media
- Observe appearance of bacteria and yeast grown on several different types of media:
nutrient agar, potato dextrose agar, sabourand dextrose agar, phenyl ethyl alcohol
agar, Mac Conkey agar and mannitol salt agar
Keywords
Streak plating • Single colony • Selective • Differentiate • Enrich • Blood agar
MacConkey agar • Mannitol salt agar • Modified Oxford Agar •XLT-4 agar.
INTRODUCTION
Culturing Microorganisms:
Microorganisms need a constant nutrient supply to survive:
- Free-living microorganisms acquire nutrients from the environment
- Parasitic microorganisms acquire nutrients from the host
Nutrients provided via artificial media:
- Liquid (broth)
- Solid (agar)
- Biochemical and nutritional requirements for microbial growth

General Growth Media

- Supports growth of many types of microorganisms (bacteria, yeast, mold, fungi)
- General growth medium is neither selective nor differential
- General growth media used in lab are Nutrient agar (NA) and tryptic Soy Agar (TSA)

Enrichment Media
- Complex media containing additives that enhance the growth of certain organisms,
for example:
o Growth factors
o Blood
o Additional vitamins or nutrients
 - Useful when the organisms which you wish to culture is present in relatively small
numbers in a mixed culture

Selective medium:
- Media that use selective agents to inhibit growth of certain microorganisms thereby
selecting, for the growth of other microorganism(s)
- Contains one or more components (chemical or antibiotics) that suppress the growth
of some microorganisms without affecting the ability of the desired organism to
grow.
- Examples of selective agents:
o High salt content
o High or low pH
o Other chemical additives
- Examples of selective media:
o PDA: Potato Dextrose Agar
o SDA: Sabouraud Dextrose Agar
o PEA: Phenylethyl Alcohol Agar
Potato Dextrose Agar (PDA):
- PDA is selective for yeast, molds and fungi
- PDA contains dextrose as a carbohydrate source which serves as a growth stimulant
and potato infusion that provides a nutrient base for luxuriant growth of most fungi.
Agar is added as the solidifying agent
- Has a lower pH (acidic) than bacterial media
- Discourages the growth of bacteria
- Yeasts and molds are more tolerant of acidic conditions
- Used for butter and dairy products

Sabouraud Dextrose Agar (SDA)

- SDA is Selective for yeasts, molds and fungi
- Has an acidic pH to inhibit bacterial growth
 - The three essential components of the SDA medium, which confer the growth of
fungi are peptone, dextrose (glucose) and pH. The mycological peptone (a mixture of
animal and plant peptones) provides a nourishing source of amino acids and
nitrogenous compounds while dextrose acts as a source of carbon and energy.
Phenylethyl Alcohol Agar (PEA)
- PEA is selective medium used for the isolation of gram positive bacteria such as
Staphylococci and Streptococci.
- Phenylethyl alcohol exert inhibitory bacteriostatic action on gram-negative bacteria
by inhibiting their DNA synthesis.
- May contains 5% sheep blood which makes it selective for gram-positive organisms.

Differential media
- Useful in grouping bacteria according to biochemical characteristics
- Contain additives (i.e. dyes or stains, etc.) that causes an observable color change
when a particular chemical reaction occurs
- Example of Differential agent is lactose:
- Fermentation decreases pH, indicator in the media causes a color change
- Identify potentially pathogenic, non-lactose fermenters (i.e. Salmonella, Shigella,
Yersinia
- Identify and enumerate coliforms: Gram negative (G-), bacillus shaped, lactose
fermenting, enteric (gut) organisms
Note that some media are BOTH selective and Differential
- Eosin Methylene Blue Agar (EMB)

Eosin Methylene Blue Agar (EMB)

- EMB is Selective for Gram negative bacteria
- Contains dyes (eosin and methylene blue) which are toxic to gram positive bacteria
- EMB is Differential for lactose fermentation
- Lactose fermenters will stain with the methylene blue dye to blue eosinate dye to a
dark purple or metallic green “sheen” colour

MacConkey Agar (Mac)

 - Mac is selective:
Gram negative organisms grow well, Gram positive organisms do not. This is due to
the addition of bile salts and crystal violet.
- Mac is Differential:
Lactose fermenters will appear pink or brick red, non lactose fermenters appear- off-
white or colorless due to the addition of indicator, neutral red
LACTOSE FERMETERS (PINK)
FERMENTING E.COLI (PINK)

FOUR TYPES OF ENTEROBACTER NON FERMENTING

PSEUDOMONAS (COLORLESS)

Mannitol Salt Agar (MSA)

- MSA is Selective for Gram positive cocci
- Contains a high salt content, which inhibits the growth of other organisms
- Helps identify Staphylococci and Micrococci
- MSA Differential for Mannitol fermentation
- Fermentation of mannitol decreases the pH below 6.8, changes the colour of the
media from bright pink to yellow
Media Selective Selects Selective Differential Differentiates Differential
Y/N for Agent Y/N Agent
Nutrient
Agar (NA)
Potato
Dextrose
Agar
(PDA)
Sabouraud
dextrose
Agar
(SDA)
Phenyl
ethyl
alcohol
Agar
(PEA)
Eosin
Methylene
Blue Agar
(EMB)
Mac
Conkey
Agar (Mac)
Mannitol
Salt Agar
(MSA)

Medium for Isolating Probiotic Bacteria

- The term 'lactic acid bacteria' ('lactics') refers to species of the following genera:
Lactobacillus, Streptococcus, Pedicoccus and Leuconostoc.
- The species included are all able to produce lactic acid in considerable amounts.
- They are Gram-positive and catalase- and oxidase-negative and are fastidious in their
nutritional requirements.
Liquid (Broth) Medium
These media contain specific amounts of nutrients but don’t have a trace of gelling agents such
as gelatin or agar. Commonly used liquid media in the lab are; nutrient broth, glucose
broth, brain-heart infusion (BHI) broth, alkaline peptone water (APW), tryptic soy broth (TSB),
and selenite F broth. Broth medium serves various purposes such as propagation of many
organisms, fermentation studies, and various other tests.
Lactic acid Bacteria on MRS-Agar
REVIEW QUESTIONS

1. Mannitol salt agar (MSA) only allows the growth of halophiles (salt-loving microbes);
non halophiles will not grow. Among the halophiles, mannitol fermenters will produce
acid that turns the pH indicator yellow; mannitol non fermenters leave the medium red.
Onto MSA you inoculate a halophilic mannitol fermenter and a halophilic mannitol non-
fermented. In this case, the medium is acting as a ______ medium(s).
A. Selective
B. Differential
C. Selective and differential
D. Enriched
2. Brief explain why Salmonella growing on XLT-4 agar is red colony with black center.
3. Phenol red is utilized in MacConkey agar to:
A. Inhibit microscopic organisms
B. Detect the production of gas by-products
C. Conduct the sulfate reduction test
D. Detect the acid reaction of lactose fermentation