MICROBIAL PHYSIOLOGY AND

METABOLISM
MIC 322
(2)
GROWTH MEDIA
Introduction
•Microorganisms need nutrients, a source of energy
and certain environmental conditions in order to
grow and reproduce. In the natural environment,
microbes have adapted to the habitats most
suitable for their needs. In the laboratory, however,
these requirements must be met by growth media
otherwise refer to as culture media.

•A culture medium is a special medium used in

microbiological laboratories to propagate different
kinds of microorganisms. Since there are many
types of microorganisms, each having unique
properties and requiring specific nutrients for
growth, there are many types of culture media
based on what nutrients they contain and what
function they play in the growth of microorganisms.
Introduction cont…
•The nutritional needs of bacteria can be met
through specialized microbiological media
that typically contain extracts of proteins (as
a source of carbon and nitrogen), inorganic
salts such as potassium phosphate or sodium
sulfate, and in some cases, carbohydrates
such as glucose or lactose. For fastidious
bacteria (meaning, those that are picky
eaters) vitamins and/or other growth factors
must be added as well.
 Types of Growth Media
Microbial culture media can be of
different type, depending on the
nutritional growth requirements of the
microorganisms. They are often
classified on the idea of:
•Consistency
•Composition
•Purpose
Classification Based on Consistency

Culture Media can be described

according to the physical state:
•Liquid
•Solid
•Semi-solid
• Biphasic
Classification Based on Consistency - Liquid
Media
• These are available for use in test-tubes, bottles or
flasks. These media do not contain any traces of
solidifying agents, such as agar or gelatin, and large
growth of bacterial colonies can be observed in the
media.
• They are used for the profuse growth of
microorganisms and fermentation studies and allow
for uniform and turbid growth of bacterial strains. The
turbidity or cloudiness of the broth is due to the light
reflected by the bacteria present in the culture.
• Liquid media are also referred to as Broth. The most
commonly used growth media for microorganisms are
nutrient broths, i.e., liquid media containing nutrients.
Classification Based on Consistency - Liquid Media
cont…
•In such broths, microorganisms usually grow
exponentially until growth is limited by either
insufficient nutrient availability or the
accumulation of growth-inhibiting substances.
Other examples include Tryptic soy broth, phenol
red carbohydrate broth and Methyl Red–Voges
Proskauer (MR-VP) broth and peptone broth
•Some of the drawbacks of culturing bacteria in
liquid media are that properties of bacteria are not
visible in liquid media and the presence of more
than one type of bacteria cannot be detected.
Solid Media
• Any liquid medium can be rendered solid by the addition of
certain solidifying agents such as gellan gum and agar agar.
Agar agar (simply called agar) is the most commonly used
solidifying agent. It is an unbranched long chain of
polysaccharide obtained from the cell membranes of some
from red algae (Rhodophyceae), such as the genera Gelidium

• Agar is composed of two polysaccharides (70% agarose and

30% agaropectin). It is used as a solidification agent because
it:
• is not hydrolyzed by microorganisms
• usually free from growth promoting or growth retarding
substances and doesn’t contribute any nutritive property
• melts at high temperatures (95OC), and yet solidifies when
the temperature falls below 45 degrees Celsius.
 Solid Media cont…
• Solid growth media is used in the following forms: agar
plates, agar slants, and agar deeps. To make agar deeps or
agar slants, melted agar is poured into a test tube and then
allowed to solidify vertically (agar deep), or at a slant (agar
slant). Agar plates are made by pouring melted agar into a
petri dish.
• Most commonly, 1.3% agar is used to prepare solid media in
labs but up to a concentration of 1.5-2.0% can be used.
Examples of solid media are blood agar, nutrient agar,
McConkey agar, and chocolate agar.
• The advantages of solid media over liquid media are that
they are used to grow microorganisms in their full physical
form, prepare bacterial pure cultures, or isolate bacteria to
study colony characteristics.
Semi-solid media
•These media have 0.2-0.5% agar concentration,
and due to the reduced agar concentration, appear
as fairly soft, jelly-like substance. They are mainly
used to study the motility of microorganisms,
distinguish between motile and non-motile
bacterial strains by cultivation in stab tubes (e.g. U-
tube and Cragie’s tube), and cultivate micro-
aerophilic bacteria.
•Bacteria grown on these media appear as thick
line. Examples of semi-solid media are: Hugh and
Leifson’s oxidation fermentation medium, Stuart’s
and Amies media, and Mannitol motility media
Biphasic Media
• Biphasic medium or diphasic medium is called so because it
contains both solid as well as the liquid medium in the same
container. And sometimes in the place of agar, egg yolk and
serum are added to the media as a solidifying agent.
• The inoculum is added to the liquid medium and when
subcultures are to be made, the bottle is simply tilted to
allow the liquid to flow over the solid medium. This obviates
the need for frequent opening of the culture bottle to
subculture.

• Biphasic medium is primarily used to avoid contamination. It

is applicable to Brucella and Mycobacterium cultures, which
are highly contagious organisms. Examples are Lowenstein
Jensen medium and Dorset egg medium, which contain egg
yolk, and Loeffler’s serum slope, which contain serum.
Classification based on the nutritional
component
1. Simple media: These are general-purpose media that
supports the growth of non-fastidious microbes, and
it is primarily used for the isolation of
microorganisms. Those bacteria that are able to grow
with minimal requirements are said to non-fastidious
and those that require extra nutrients are said to be
fastidious. Examples are nutrient broth, peptone
water, and nutrient agar.
2. Complex media: These are media containing
nutrients in unknown quantities that are added to
bring about a particular characteristic of a microbial
strain. Examples are tryptic soy broth, blood agar, and
nutrient broth.
Classification based on the nutritional
component cont…
3. Synthetic media (or defined media):
•Synthetic medium is a type of chemically defined
media and is produced from pure chemical
substances. A defined medium has well known
concentration of ingredients, like sugar (glucose or
glycerol) and nitrogen source (such as ammonium
salt or nitrate as inorganic nitrogen).
•It is generally used in scientific research to study
the physiology, metabolism and nutritional
requirements of certain microorganisms. An
example is Czapek Dox Medium.
Classification based on the nutritional
component - Synthetic media cont…
•They are of two types: simple and complex.
oSimple Synthetic Media: Simple synthetic
media contain a carbon and energy source, an
organic source of nitrogen and various inorganic
salts in buffered aqueous solution.
oComplex Synthetic Media: This media contains
all the ingredients of simple synthetic media. In
addition, it also contains amino acids, purines,
pyrimidines and many other growth factors
depending upon the nature and growth
requirements of the organism
Classification based on functional use or
application
i. GENERAL-PURPOSE MEDIA
• General-purpose media (also known as basic or complex media)
are non-selective routine culture media that microbiology
laboratories. They have carbon and nitrogen sources that boost
the growth of a broad spectrum of microorganisms.
• They contain complex organic ingredients such as yeast, animal,
or plant material that are not chemically precisely determined,
so they vary slightly in composition from batch to batch.
• These media are used to culture non-fastidious microorganisms
that do not need extra growth nutrients. Commonly used
general-purpose media in the microbiological laboratory include
nutrient agar and broth, peptone water, as well as tryptic soy
broth and agar, Potato dextrose agar (PDA)
Classification based on functional use or application
cont…
ii. DIFFERENTIAL OR INDICATOR MEDIA
• Differential media (or indicator media) are so called because they
have indicative properties and use the biochemical characteristics of
a microorganism. . Molecular biologists utilize this sort of media to
identify recombinant bacterium strains.
• They contain certain indicators like dyes or metabolic substrates in
the medium composition that lead colonies of certain organisms to
take on a distinctive appearance when they utilize or react with
these components.
• Some differential media contain pH indicators such as neutral red,
phenol red and methylene blue. Change in colour occurs if a
bacterial species produces acid during growth that lowers the pH of
the medium. This approach is used in MacConkey agar, CLED
(cystine–lactose–electrolyte-deficient) agar, TCBS (Thiosulfate –
citrate–bile salts–sucrose) agar and XLD (Xylose lysine deoxycholate)
agar.
Classification based on functional use or
application cont…
• MacConkey agar is the most commonly used medium to
culture and identify Gram negative bacilli (especially
enterobacteriaceae members). It contains bile salts (selective
agent), lactose (sugar), peptone and neutral red (pH indicator),
agar and water. Those bacteria that can ferment lactose
produce pink coloured colonies where non-lactose fermenting
colonies produce colourless colonies

• Another example is Blood agar: In blood agar, three types of

blood cell lysis or heamolysis are observed: alpha, beta, and
gamma hemolysis. The agar allows the growth of many
microorganisms, however, their ability to lyse blood cells
differs, and this helps to distinguish the bacterial colonies.
Classification based on functional use or
application cont…
iii. Selective media
• Selective media in microbiology are particularly useful
where the desired organism is only one among many in the
inoculum.
• Selectivity is achieved by composing the media to be
optimal for the growth of the desired microorganisms
and/or unfavorable for other organisms. This can include
adding antibiotics, high salt concentrations, low pH or
inhibitors like bile salts.
• An example of selective media is mannitol salt agar. It
contains a high concentration of sodium chloride that
inhibits the growth of most organisms but permits
staphylococci to grow.
Common selective media and the organism the
support its growth
Culture media
Salmonella-Shigella Agar
Pseudosel Agar (cetrimide
agar)
Thiosulfate citrate bile salts
sucrose (TCBS) agar
Crystal Violet Blood Agar
Thayer Martin Agar
Birdseed agar
Inhibiting substances Bacteria
Contains bile salts, brilliant Used for the isolation of
green, and sodium citrate Salmonella
Contains cetrimide (antiseptic Used to recover Pseudomonas
agent) aeruginosa
Have elevated pH of about Used for isolating Vibrio
8.5-8.6 cholerae
Contains 0.0002% crystal Used for Streptococcus
violet pyogenes
Contains antibiotics; Used for Neisseria
vancomycin, colistin, and gonorrhoeae
nystatin
Contains Guizotia abyssinica Cryptococcus neoformans
seeds, glucose, creatinine,
chloramphenicol,
monopotassium phosphate
Classification based on functional use or application cont…
iv. Enrichment Media
•Enrichment media are for liquid cultivation and
favour the growth of a particular
microorganism over others in a sample.
•Enrichment is often performed to have enough
organism or cells available for subsequent
detection, isolation and / or identification.
•Examples are selenite F broth which is used to
isolate Salmonella typhi from a fecal sample,
tetrathionate broth, and alkaline peptone
water.
Classification based on functional use or
application cont…
v. Enriched Media
•Enriched media, on the other hand, are semi-
selective: they contain supplements such as
blood, serum, and egg yolk. The addition of such
substances can support the growth of those
bacteria which are comparatively exacting in their
demands.
•Examples are chocolate agar, blood agar, and
Loeffler’s serum slope. Chocolate media is used to
grow N. gonorrheae while blood agar (which is
prepared by adding 5-10% blood by volume to a
blood agar base) is used to identify heamolytic
bacteria
Classification based on functional use or
application cont…
vi. TRANSPORT AND STORAGE MEDIA
•Transport media are useful for clinical
specimens which are required to be
transferred immediately to labs to maintain
the viability of potential pathogens and to
prevent overgrowth of commensals or
contaminating microorganisms.
•Therefore, most transport media lack the
usual ingredients of culture media and
contain only substances that do not promote
microbial growth.
Classification based on functional use or
application (Transport Media) cont…
• Some of them are semi-solid in consistency, and examples
include:
• Sach’s buffered glycerol saline: It’s used to transport feces
from patients suspected to be suffering from bacillary
dysentery.
• Cary Blair transport and Venkatraman Ramakrishnan
media: Fecal samples collected from suspected cholera
patients are transported using these media.
• Pike’s medium: A throat specimen containing Streptococci
is transported using this medium
• Microorganisms may also be need to be stored for a long time.
The sole purpose of storage media is to store and preserve the
culture in laboratories. Examples: Egg saline medium, chalk
cooked meat broth
Classification based on functional use or application
cont…
vii.Anaerobic media
•These media are for anaerobic bacteria which
require low oxygen levels, extra nutrients, and
reduced oxidation-reduction potential.
•It is supplemented with heamin and vitamin K
nutrients and oxygen is removed by boiling it in
a water bath and sealing it with paraffin film.
Examples are: Thioglycollate broth and
Robertson Cooked Meat (RCM) medium which
is commonly used to grow Clostridium spp.