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Iraqia university / College of medicine

Microbiology Dept.
T.A. Yasen Alani
 Bacteria have to be grown (cultured) for
them to be identified.
 By appropriate procedures they have to be
grown separately (isolated) on culture
media and obtained as pure for study.
Colony – macroscopically visible collection
of millions of bacteria originating from a
single bacterial cell.
 Used for preparing solid medium
 Obtained from seaweeds.
 No nutritive value
 Not affected by the growth of the bacteria.
 Melts at 98oC & sets at 42oC
 2% agar is employed in solid medium
Growth Media
 Bacteria and other microbes have
particular requirements for growth.

 In order to successfully grow bacteria

in lab, we must provide an environment
suitable for growth.

 Growth media (singular = medium) are used to

cultivate microbial growth.

 Media = mixtures of nutrients that the microbes need to live.

Also provides a surface and the necessary moisture and pH to support
microbial growth.

 Tryptic Soy Agar (TSY) is the medium that we most often use. Complex
nutrient media which supports the growth of a wide variety of microbes.
Types of culture media
I. Based on their consistency
a) solid medium
b) liquid medium
c) semisolid medium
II. Based on the constituents/ ingredients
a) simple medium
b) complex medium
c) synthetic or defined medium
d) Special media
Special media
– Enriched media
– Enrichment media
– Selective media
– Indicator media
– Differential media

III.Based on Oxygen requirement

- Aerobic media
- Anaerobic media
Solid media – contains 2% agar
 Colony morphology, pigmentation, hemolysis can
be appreciated.
 Eg: Nutrient agar, Blood agar

Liquid media – no agar.

 For inoculum preparation, Blood culture, for the
isolation of pathogens from a mixture.
 Eg: Nutrient broth

Semi solid medium – 0.5% agar.

 Eg: Motility medium
Simple media / basal media
- Eg: NB, NA
- NB consists of peptone, meat extract, NaCl,
- NB + 2% agar = Nutrient agar

Enriched media: selects for a certain group of

 Substances like blood, serum, egg are
added to the basal medium.
 Used to grow bacteria that are exacting in
their nutritional needs.
 Eg: Blood agar, Chocolate agar

Q: What does selective mean?

Q: What does differential mean?

Specialized Media:

McConkey’s, Mannitol
Salt & Blood Agar

Look at the plates on your lab bench that are red/pink in color.

McConkey’s = lighter, purplish-pink

Mannitol Salt = orangish-pink
Blood Agar = very dark red

Unlike TSY media, these specialized selective & differential media plates are already
prepared for you.
MacConkey's (MAC)

MacConkey’s media is both selective & differential.

1. Selective because it only grows Gram-negative

bacteria. Inhibits the growth of Gram-positive

2. Differential because neutral red (pH-sensitive

dye) and lactose (type of sugar) have been added
to media.
- Bacteria that use lactose for food (lactose fermenters),
produce acidic
metabolites that trigger the pH sensitive dye to turn pink.
- So lactose fermenting bacteria will grow in bright pink
colonies while
non-lactose fermenters will be colorless and clear.

** Enteric bacteria are the most frequently encountered

bacteria isolated from many types of clinical specimens.
They are most commonly lactose fermenters.
Mannitol Salt (MSA)

Mannitol Salt media is both selective &


1. Selective because it has a high NaCl (7.5%)

concentration, and few types of bacteria can
grow on this hypertonic medium.
Members of genus Staphylococcus are
halophilic, and grow well on this media.

2. Differential because this medium contains a pH-

sensitive dye to identify organisms that ferment
mannitol. Organic acids wastes mannitol
fermenters produce change the medium from red
to yellow.
MSA works well for identifying pathogenic
staphylococci, such as Staphylococcus aureus,
which will ferment mannitol.
Most non-pathogenic staphylococci
(Staphylococcus epidermidis) will not ferment
** Gram –ve inhibited growth.
Blood agar (BAP)
Most specimens received in a clinical microbiology lab are plated
onto Blood Agar. It is an enriched medium that will grow
even fastidious bacteria.
Also contains 5% sheep blood.
This media is not selective. It is enriched and differential:

Certain bacteria produce enzymes (hemolysins…say hemo-lice-ins)

that act on red cells to produce either:
* Beta hemolysis: Enzymes lyse the blood cells completely,
producing a clear area around the colony.
* Alpha hemolysis: Incomplete hemolysis produces a greenish
discoloration around the colony.
* Gamma hemolysis: No effect on the red cells.

Blood agar is usually inoculated from a patient’s throat swab.

Microbiologist are trying to detect Group A beta hemolytic

Streptococcus pyogenes (a Gram-positive cocci-shaped bacteria
that causes Beta hemolysis on blood agar.)

Normal flora of the throat will exhibit alpha or gamma

Blood agar Chocolate agar

Used for the growth of Gram positive cocci and fastidious organisms
like Neisseria & Haemophilus species.
Enrichment media: selects for one
 Liquid media used to isolate
pathogens from a mixed culture.
 Media is incorporated with inhibitory
substances to suppress the unwanted

 Eg:
– Selenite F Broth – for the isolation of
Salmonella, Shigella
Selective media
 The inhibitory substance is added to a solid
 Mac Conkey’s medium for gram negative
 TCBS – for V.cholerae
 LJ medium – M.tuberculosis
 Wilson and Blair medium – S.typhi
 Potassium tellurite medium – Diphtheria
Complex media
 Media other than basal media.
 They have added ingredients.
 Provide special nutrients

Synthetic or defined media

 Media prepared from pure chemical
substances and its exact composition is
Mac Conkey’s medium TCBS
Potassium Tellurite media LJ media
Indicator media
 These media contain an indicator which
changes its colour when a bacterium
grows in them.
 Eg:
– Blood agar
– Mac Conkey’s medium
– Christensen’s urease medium
Urease medium
Differential media
 A media which has substances
incorporated in it enabling it to distinguish
between bacteria.
 Eg: Mac Conkey’s medium
– Peptone
– Lactose
– Agar
– Neutral red
– Taurocholate
 Distinguish between lactose fermenters &
non lactose fermenters.
 Lactose fermenters – Pink colonies
 Non lactose fermenters – colourless colonies