Lecture 03

04/08/2018

Types of Media for growth of

microorganisms
 Media: The Foundations
of Culturing

• The development of techniques for growing microbes enabled the

close examination of a microbe and its morphology, physiology and
genetics.

• Basic principle for a medium:

The microorganisms being cultured need to be provided with all of their
required nutrients in an artificial medium.

• Culture media are contained in test tubes, flasks or Petri dishes.

• Inoculation is performed by tools as loops, needles, pipettes, and
swabs.
• For an experiment to be properly controlled,
sterile technique is necessary.
• Sterile technique: Inoculation with a sterile
medium and inoculating tools with sterile tips
must be used.
• Measures must be taken to prevent introduction
of non-sterile materials, such as room air, fingers
and any other thing, directly into the media.
 Types of Media
• Most media discussed are designed for bacteria
and fungi, though algae and some protozoa can
be propagated in media.

• Viruses can only be cultivated in live host cells.

Media fall into three general categories based
on their properties: physical state, chemical
composition, and functional type.
 Physical States of Media

• Liquid media are defined as water-based solutions that do not

solidify at temperatures above freezing and that tend to flow
freely when the container is tilted.
• These media, termed broths, milks, or infusions, are made by
dissolving various solutes in distilled water.
• Growth occurs throughout the container and can then present a
dispersed, cloudy, or flaky appearance.
• A common laboratory medium, nutrient broth,
contains beef extract and peptone dissolved in
water.
• Methylene blue milk and litmus milk contain
whole milk and dyes.
• Fluid thioglycollate is a slightly viscous broth
used for determining patterns of growth in
oxygen.
• Urea broth is used to show a biochemical reaction
in which the enzyme urease digests urea and
releases ammonium.
• This raises the pH of the solution and causes the
dye to become increasingly pink.
 Left: uninoculated broth, pH 7;
 middle: growth with no change;
 right: positive, pH 8.0.
• Presence-Absence Broth is for detecting the
presence of coliform bacteria in water samples.
• It contains lactose and bromcresol purple dye.
• As coliforms use lactose, they release acidic
substances. This lowers the pH and changes the dye
from purple to yellow (right is Escherichia coli).
• Noncoliforms such as Pseudomonas grow but do not
change the pH (purple color indicates neutral pH).
 Semisolid media
• At ordinary room temperature, semisolid media
exhibit a clotlike consistency because they contain an
amount of solidifying agent (agar or gelatin) that
thickens them but does not produce a firm substrate.
• Semisolid media are used to determine the motility
of bacteria and to localize a reaction at a specific site.
• Motility test medium and sulfur indole motility
(SIM) medium both contain only 0.3–0.5% of agar.
• In both cases, the medium is stabbed carefully in the
center with an inoculating needle and later observed
for the pattern of growth around the stab line.
• Semisolid media have more body than liquid
media but are softer than solid media.
• They do not flow freely and have a soft,
clotlike consistency.
• SIM medium:
(1) An uninoculated tube. The location of growth can
be used to determine
(2) Nonmotility
(3) or motility
(4) The medium reacts with any H2S gas to produce a
black precipitate.
 Solid media
• Solid media provide a firm surface on which
cells can form discrete colonies and are
advantageous for isolating and culturing
bacteria and fungi.
 Liquefiable solid media
• Liquefiable solid media, or reversible solid media, contain a solidifying
agent that changes its physical properties in response to temperature.
• The most widely used and effective of these agents is agar, a
polysaccharide isolated from the red alga Gelidium.
• The benefts of agar are numerous.
• It is solid at room temperature, and it melts (liquefies) at the boiling
temperature of water (100°C or 212°F).
• Once liquefied, agar does not resolidify until it cools to 42°C (108°F),
so it can be inoculated and poured in liquid form at temperatures
(45°C to 50°C) that will not harm the microbes or the handler (body
temperature is about 37°C or 98.6°F).
• Agar is flexible and moldable, and it provides a basic
framework to hold moisture and nutrients.
• Another useful property is that it is not readily digestible and
thus not a nutrient for most microorganisms.
• Any medium containing 1% to 5% agar usually has the word
agar in its name.
• Nutrient agar is a common one. Like nutrient broth, it
contains beef extract and peptone, as well as 1.5% agar by
weight.
 Gelatin
• Gelatin creates a reasonably solid surface in
concentrations of 10% to 15%.
• The main drawback for gelatin is that it can be
digested by microbes and will melt at room
and warmer temperatures, leaving a liquid.
 Nonliquefiable solid media
• Nonliquefiable solid media do not melt. They
include materials such as rice grains (used to
grow fungi), cooked meat media (good for
anaerobes), and egg or serum media that are
permanently coagulated or hardened by moist
heat.
 Chemical Content of Media
• Media with a chemically defined composition are termed
synthetic.
• Such media contain pure chemical nutrients that vary little
from one source to another and have a molecular content
specified by an exact formula.
• Synthetic media come in many forms. Some media, such as
minimal media for fungi, contain a few salts and amino acids
dissolved in water.
• Others contain dozens of precisely measured ingredients.
• But they can only be used when the exact nutritional needs of
the test organisms are known.
• A defined medium that was developed to grow the parasitic
protozoan Leishmania required 75 different chemicals.
 Nonsynthetic, or Complex medium
• If even one component of a given medium is not chemically definable, the
medium is a nonsynthetic, or complex medium.
• The composition of this type of medium is not definable by an exact
chemical formula.
• Substances that can make it nonsynthetic are extracts from animal or
plant tissues including such materials as ground-up cells and secretions.
• Other examples are blood, serum, meat extracts, infusions, milk, soybean
digests and peptone.
• Peptone is a partially digested protein, rich in amino acids, that is often
used as a carbon and nitrogen source.
• Nutrient broth, blood agar, and MacConkey agar, though different in
function and appearance, are all complex nonsynthetic media.
• They present a rich mixture of nutrients for microbes with complex
nutritional needs.
 General-purpose media
• General-purpose media are designed to grow a broad spectrum of
microbes that do not have special growth requirements.
• As a rule, these media are nonsynthetic (complex) and contain a
mixture of nutrients that could support the growth of a variety of
bacteria and fungi.
• These include nutrient agar and broth, brain-heart infusion, and
trypticase soy agar (TSA).
• TSA is a complex medium that contains partially digested milk
protein (casein), soybean digest, NaCl, and agar.
 Enriched medium
• An enriched medium contains complex organic substances such
as blood, serum, hemoglobin, or special growth factors that
certain species must be provided in order to grow.
• These growth factors are organic compounds such as vitamins
and amino acids that the
microbes cannot synthesize themselves.
• Bacteria that require growth factors and complex nutrients are
termed fastidious.
• Blood agar is made by adding sterile animal blood (usually from
sheep) to a sterile agar base, is widely employed to grow
fastidious streptococci and other pathogens.
• Blood agar plate growing bacteria from the human
throat.
• Note that this medium can also differentiate among
colonies by the zones of hemolysis they may show.
• Note that some colonies have clear zones
(beta hemolysis) and others have less defined zones.
• Culture of Neisseria sp. on Thayer-Martin
medium or chocolate agar.
• chocolate agar is made by heating blood agar
and does not contain chocolate—it just has
that appearance.
• It does not produce hemolysis.
 Selective and Differential Media
• Some of the cleverest and most inventive media
recipes belong to the categories of selective and
differential media.
• These media are designed for special microbial
groups, and they have extensive applications in
isolation and identification.
• They can permit, in a single step, the preliminary
identification of a genus or even a species.
 Selective medium
• A selective medium contains one or more agents that inhibit the
growth of a certain microbe or microbes (call them A, B, and C)
but not another (D).
• This difference favors, or selects, microbe D and allows it to grow
by itself.
• Selective media are very important in primary isolation of a
specific type of microorganism from samples containing mixtures
of different species—for example, feces, saliva, skin, water, and
soil.
• They suppress the unwanted background organisms and allow
growth of the desired ones.
• Mannitol salt agar (MSA) contains a high concentration of NaCl (7.5%)
that is quite inhibitory to most human pathogens.
• One exception is the genus Staphylococcus, which grows well in this
medium and consequently can be amplified in mixed samples.
• It is also differential because it contains a dye (phenol red) that changes
color under variations in pH, and mannitol, a sugar that can be converted
to acid.
• The left side shows S. epidermidis, a species that does not use mannitol
(red).
• The right shows S. aureus, a pathogen that uses mannitol (yellow).
• Bile salts, a component of feces, inhibit most gram-positive
bacteria while permitting many gram-negative rods to grow.
• Media for isolating intestinal pathogens (MacConkey agar,
Hektoen enteric [HE] agar) contain bile salts as a selective
agent.
• MacConkey agar differentiates between lactose-fermenting
bacteria (indicated by a pink-red reaction in the center of the
colony) and lactose-negative bacteria (indicated by an off-
white colony with no dye reaction).
 Other selective agents
• Dyes such as methylene blue and crystal violet also
inhibit certain gram-positive bacteria.
• Other agents that have selective properties are
antimicrobial drugs and acid.
• Some selective media contain strongly inhibitory
agents to favor the growth of a pathogen that would
otherwise be overlooked because of its low numbers
in a specimen.
• Selenite and brilliant green dye are used in media to
isolate Salmonella from feces.
• Sodium azide is used to isolate enterococci from
water and food.
 Differential media
• Differential media grow several types of
microorganisms but are designed to bring out
visible differences among those
microorganisms.
• Differences show up as variations in colony
size or color, in media color changes, or in the
formation of gas bubbles and precipitates
• Several newer forms of differential media
contain artificial substrates called chromogens
that release a wide variety of colors, each tied
to a specific microbe.
• Other chromogenic agar is available for
identifying Staphylococcus, Listeria, and
pathogenic yeasts.
• Triple sugar iron agar (TSIA) inoculated on the surface and
stabbed into the thicker region at the bottom (butt).
• This medium contains three sugars, phenol red dye to indicate
pH changes (bright yellow is acid, various shades of red,
basic), and iron salt to show H2S gas production. Reactions are
(1) no growth;
• (2) growth with no acid production (sugars not used);
• (3) acid production in the butt only;
• (4) acid production in all areas of the medium;
• (5) acid and H2S production in butt (black precipitate).
• A medium developed for culturing and identifying
the most common urinary pathogens.
• CHROMagar Orientation™ uses color-forming reactions to
distinguish at least seven species and
permits rapid identification and treatment. In the example,
the bacteria were streaked so as to spell their own names.
 Dyes as differential agents
• Dyes are effective differential agents because many of them
are pH indicators that change color in response to the
production of
an acid or a base.
• For example, MacConkey agar contains neutral red, a dye that
is yellow when neutral and pink or red when acidic.
• A common intestinal bacterium such as Escherichia coli that
gives off acid when it metabolizes the lactose in the medium
develops red to pink colonies, and one such as Salmonella that
does not give off acid remains its natural color (off-white).
 Carbohydrate fermentation in broths.
• This medium is designed to show
fermentation (acid production)
using phenol red broth and gas
formation by means of a small,
inverted Durham tube for
collecting gas bubbles.
• The tube on the left is an uninoculated
negative control;
• the center tube is positive for acid (yellow) and
gas (open space);
• the tube on the right shows growth but neither
acid nor gas.
 Miscellaneous Media
• A reducing medium contains a substance (thioglycollic acid
or cystine) that absorbs oxygen or slows the penetration of
oxygen in a medium, thus reducing its availability.
• Reducing media are important for growing anaerobic
bacteria or for determining oxygen requirements of isolates.
• Carbohydrate fermentation media contain sugars that can be
fermented (converted to acids) and a pH indicator to show
this reaction.
 Transport media
• Transport media are used to maintain and
preserve specimens that have to be held for a
period of time before clinical analysis or to
sustain delicate species that die rapidly if not
held under stable conditions.
• Stuart’s and Amie’s transport media contain
buffers and absorbants to prevent cell
destruction but will not support growth.
• Assay media are used by technologists to test the
effectiveness of antimicrobial drugs and by drug
manufacturers to assess the effect of disinfectants,
antiseptics, cosmetics, and preservatives on the growth of
microorganisms.

• Enumeration media are used by industrial and

environmental microbiologists to count the numbers of
organisms in milk, water, food, soil, and other samples.
 Animal cell culture
• A number of significant microbial groups (viruses, rickettsias,
and a few bacteria) will only grow on live cells or animals.
• These obligate parasites have unique requirements that must be provided by
living animals such as rabbits, guinea pigs, mice, chickens, and the early life
stages (embryos) of birds.
• Such animals can be an indispensable aid for studying, growing, and identifying
microorganisms.
• Animal inoculation is an essential step in testing the effects of
drugs and the effectiveness of vaccines before they are administered to humans.
• Animals are an important source of antibodies, antisera, antitoxins, and other
immune products that can be used in
therapy or testing.