question is: What is culture media?
Describe the various
methods of culture media.
➡️ classification based on nutritional requirement
a) chemically defined media
b) complex media.
c) anaerobic media
d) selective/differential media.
e) special media
f) enrichment media.
Detailed Version
A culture medium is a nutrient material that is prepared in a laboratory to grow
microorganisms. The microorganisms that grow and multiply in or on this medium are
called a culture. To grow a specific microorganism, the culture medium must contain
the right nutrients, sufficient moisture, a properly adjusted pH, and a suitable oxygen
level (or none at all). It must also be sterile initially and incubated at the correct
temperature. Agar, a complex polysaccharide from marine algae, is commonly added to
media to solidify it for growing bacteria on a solid surface. Agar is valuable because few
microbes can degrade it, and it liquefies at about 100°C but remains liquid until about
40°C, which does not harm most bacteria when poured over them.
Here are various methods of culture media, often classified based on their nutritional
requirements and intended use:
a) Chemically Defined Media
A chemically defined medium is a culture medium whose exact chemical composition
is known. To support microbial growth, this medium must provide an energy source, as
well as sources of carbon, nitrogen, sulfur, phosphorus, and any organic growth factors
the organism cannot synthesize on its own. For chemoheterotrophs, a chemically
defined medium includes organic growth factors that serve as sources of carbon and
energy, such as glucose for Escherichia coli. Organisms that require many growth
factors are referred to as fastidious. These media are typically used for laboratory
experimental work or for growing autotrophic bacteria.
Table 6.2: A Chemically Defined Medium for Growing a Typical
Chemoheterotroph, Such as Escherichia coli
Constituent Amount
Glucose 5.0 g
Ammonium phosphate, monobasic (NH4H2PO4) 1.0 g
Sodium chloride (NaCl) 5.0 g
Magnesium sulfate (MgSO4.7H2O) 0.2 g
Potassium phosphate, dibasic (K2HPO4) 1.0 g
Water 1 liter
Table 6.3: Defined Culture Medium for Leuconostoc mesenteroides
Category Constituents and Amounts
Carbon and Glucose, 25 g
Energy
Salts NH4Cl, 3.0 g; K2HPO4*, 0.6 g; KH2PO4*, 0.6 g; MgSO4, 0.1 g
Amino Acids 100–200 μg each of Alanine, arginine, asparagine, aspartate,
cysteine, glutamate, glutamine, glycine, histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, valine
Purines and 10 mg of each of Adenine, guanine, uracil, xanthine
Pyrimidines
Vitamins 0.01–1 mg each of Biotin, folate, nicotinic acid, pyridoxal,
pyridoxamine, pyridoxine, riboflavin, thiamine, pantothenate,
p-aminobenzoic acid
Trace Elements 2–10 μg each of Fe, Co, Mn, Zn, Cu, Ni, Mo
Buffer, pH 7 Sodium acetate, 25 g
Solvent Distilled Water, 1,000 ml
Notes *Also serves as buffer.
b) Complex Media
A complex medium is one where the exact chemical composition varies slightly from
one batch to another. These media are made from nutrients such as extracts from
yeasts, meat, or plants, or from digests of proteins from these and other sources. In
complex media, the energy, carbon, nitrogen, and sulfur requirements for microbial
growth are primarily met by protein. Since proteins are large and relatively insoluble,
they are partially digested by acids or enzymes into shorter chains of amino acids called
peptones, which are smaller, soluble fragments digestible by most bacteria. Vitamins
and other organic growth factors are supplied by meat or yeast extracts, with yeast
extracts being particularly rich in B vitamins. If a complex medium is in liquid form, it's
called nutrient broth; when agar is added, it becomes nutrient agar.
Table 6.4: Composition of Nutrient Agar, a Complex Medium for the Growth of
Heterotrophic Bacteria
Constituent Amount
Peptone (partially digested protein) 5.0 g
Beef extract 3.0 g
Sodium chloride 8.0 g
Agar 15.0 g
Water 1 liter
c) Anaerobic Media
Cultivating anaerobic bacteria presents a challenge because these microbes can be
killed by exposure to oxygen. To address this, special media called reducing media are
used. These media contain ingredients, such as sodium thioglycolate, that chemically
combine with and remove dissolved oxygen from the culture medium. For routine
growth of obligate anaerobes, reducing media are stored in ordinary, tightly capped test
tubes and are heated just before use to drive off any absorbed oxygen.
When growing anaerobic cultures on Petri plates to observe individual colonies, several
methods are available:
● Anaerobic jars: These sealed containers use chemical packets that produce
hydrogen and carbon dioxide. In the presence of a catalyst, hydrogen combines
with atmospheric oxygen to form water, thus removing oxygen from the jar. An
anaerobic indicator like methylene blue is often included; it is blue when oxygen
is present and colorless when oxygen is removed. There is a figure on page 204
(Figure 6.6) that illustrates an anaerobic jar.
● Anaerobic chambers: These are chambers filled with inert gases (typically
about 85% nitrogen, 10% hydrogen, and 5% carbon dioxide) and are equipped
with air locks for introducing cultures and materials. There is a figure on page
203 (Figure 6.7) that illustrates an anaerobic chamber.
● OxyPlates: These are individual Petri plates that become anaerobic chambers
through the use of an enzyme called oxyrase in the medium, which combines
oxygen with hydrogen to form water, removing oxygen.
d) Selective and Differential Media
These media are frequently used in clinical and public health microbiology to detect
specific microorganisms.
● Selective media are designed to suppress the growth of unwanted bacteria and
encourage the growth of the desired microbes. For example, bismuth sulfite agar
is used to isolate Salmonella typhi from feces, as it inhibits gram-positive bacteria
and most other gram-negative intestinal bacteria. Sabouraud's dextrose agar,
with a pH of 5.6, selectively isolates fungi because they outgrow most bacteria at
this pH.
● Differential media make it easier to distinguish colonies of the desired organism
from other colonies growing on the same plate, or they show identifiable
reactions with pure cultures in tubes or plates. For instance, blood agar is used to
identify bacterial species that destroy red blood cells; Streptococcus pyogenes,
which causes strep throat, produces a clear ring (beta-hemolysis) around its
colonies by lysing the surrounding blood cells. There is a figure on page 209
(Figure 6.9) that illustrates blood agar.
● Some media combine both selective and differential characteristics. An example
is mannitol salt agar, which contains 7.5% sodium chloride to select for
organisms tolerant to high salt concentrations, such as Staphylococcus aureus. It
also includes a pH indicator that changes color if the mannitol in the medium is
fermented to acid, thus differentiating S. aureus colonies from those that do not
ferment mannitol. There is a figure on page 212 (Figure 6.10) that illustrates
differential media (mannitol salt agar).
Table 6.5: Culture Media (Summary of Types and Purposes)
Type Purpose
Chemically Growth of chemoautotrophs and photoautotrophs; microbiological
Defined assays
Complex Growth of most chemoheterotrophic organisms
Reducing Growth of obligate anaerobes
Selective Suppression of unwanted microbes; encouraging desired
microbes
Differential Differentiation of colonies of desired microbes from others
Enrichment Similar to selective media but designed to increase numbers of
desired microbes to detectable levels
e) Special Media/Culture Techniques
Many bacteria cannot be grown on artificial laboratory media and require special
culture techniques.
● Some parasitic and fastidious bacteria must be cultured in living animals or cell
cultures. For example, Mycobacterium leprae (leprosy bacillus) is typically
grown in armadillos, and obligate intracellular bacteria like rickettsias and
chlamydias can only reproduce in living host cells.
● For aerobic bacteria that require higher or lower concentrations of CO2 than
found in the atmosphere, CO2 incubators or simple candle jars can be used.
Capnophiles are microbes that grow better at high CO2 concentrations. Candle
jars achieve a lowered oxygen concentration (about 17%) and an elevated CO2
concentration (about 3%), resembling conditions in certain body tissues where
pathogens grow.
● For dangerous microorganisms, extraordinary containment systems known as
biosafety levels (BSL) are used, ranging from BSL-1 for basic labs to BSL-4 for
the most hazardous pathogens, which involve sealed environments, filtered air,
and specialized suits.
f) Enrichment Media
An enrichment culture is used when bacteria are present in small numbers in a
sample and might be overlooked due to the presence of other, more numerous bacteria.
The enrichment medium is typically liquid and provides nutrients and environmental
conditions that specifically favor the growth of a particular microbe while discouraging
others. In essence, it acts as a selective medium, but its primary goal is to increase the
population of the desired organism to a detectable level. For example, to isolate a
microbe that can metabolize phenol from a soil sample, the sample can be placed in a
liquid enrichment medium where phenol is the only carbon and energy source. After
incubation, small amounts of this culture are transferred to fresh medium, repeatedly
diluting out non-phenol-metabolizing microbes and enriching for the desired species.
This technique is notable because phenol is normally lethal to most bacteria.
On the basis of physical status
The physical status of a culture medium primarily depends on the presence and
concentration of agar.
a) Liquid (Broth) Liquid media, often called broths, do not contain a solidifying agent
like agar. These are used for growing large numbers of bacteria and for certain
biochemical tests. For example, a complex medium in liquid form is called nutrient
broth.
b) Solid Solid media contain a solidifying agent, most commonly agar, to provide a
solid surface for microbial growth. Agar is a complex polysaccharide derived from
marine algae. It is valuable in microbiology because few microbes can degrade it, it
liquefies at about 100°C, and remains liquid until it cools to about 40°C, which allows it
to be poured over bacteria without harming them. Once solidified, it can be incubated at
temperatures approaching 100°C before reliquefying. Solid media are often contained in
test tubes (as slants for large surface area or deeps for vertical growth) or Petri
dishes (called Petri plates or culture plates). There is a figure (Figure 6.17) on page
251 that illustrates methods of preparing plates for plate counts, including pouring
melted agar.
Easy Version
A culture medium is a nutrient material made in the lab to grow microorganisms.
The microbes that grow in or on this medium are called a culture.
For growth, the medium must have:
● proper nutrients,
● enough moisture,
● correct pH,
● suitable oxygen level (or no oxygen for anaerobes),
● and it must be sterile before use.
Often agar (a substance from seaweed) is added to make the medium solid. Agar is
useful because most microbes cannot break it down, and it melts at 100°C but solidifies
at 40°C.
The different types of culture media are:
a) Chemically Defined Media
● Exact chemical composition is known.
● Provides carbon, nitrogen, sulfur, phosphorus, and energy source (e.g., glucose
for E. coli).
● Used for research or growing autotrophic bacteria.
● Organisms needing many growth factors (fastidious) are grown here.
b) Complex Media
● Exact chemical composition is not known (changes slightly in each batch).
● Made from yeast extract, meat extract, or digested proteins (called peptones).
● Provides carbon, nitrogen, sulfur, and vitamins.
● Nutrient broth (liquid) and nutrient agar (solid) are examples.
c) Anaerobic Media
● Used for microbes that cannot tolerate oxygen (obligate anaerobes).
● Contain chemicals like sodium thioglycolate to remove oxygen.
● Methods:
○ Anaerobic jar → oxygen removed by chemical reactions.
○ Anaerobic chamber → filled with gases like nitrogen and hydrogen, no
oxygen.
d) Selective and Differential Media
● Selective media → encourage desired microbes, suppress unwanted ones.
○ Example: Bismuth sulfite agar (for Salmonella typhi), Sabouraud’s agar
(for fungi).
● Differential media → show differences between microbes by visible changes.
○ Example: Blood agar (shows clear zone if red blood cells are destroyed),
○ Mannitol salt agar (selective for Staphylococcus, also changes color if
sugar mannitol is fermented).
e) Special Media / Techniques
● Some microbes cannot grow on normal media, they need special conditions:
○ Mycobacterium leprae → grown in armadillos.
○ Rickettsias and chlamydias → need living host cells.
○ Capnophiles (need high CO₂) → grown in CO₂ incubators or candle jars.
● Dangerous microbes require special labs called biosafety levels (BSL).
f) Enrichment Media
● Used when the microbe is in very small numbers and may be missed.
● Liquid media that provide special nutrients for the target microbe, while
discouraging others.
● Example: A soil sample placed in a medium with phenol as the only carbon
source → only microbes that can use phenol will grow.
On the Basis of Physical Status
The physical form of culture media depends mainly on whether agar (a solidifying
agent) is present or not.
a) Liquid (Broth):
● Does not contain agar.
● Used to grow large numbers of bacteria.
● Also used in biochemical tests.
● Example: Nutrient broth.
b) Solid:
● Contains agar to make a solid surface for bacterial growth.
● Agar comes from seaweed and is very useful because:
○ Most microbes cannot break it down.
○ Melts at 100°C, stays liquid until 40°C → can be poured safely.
○ Once solidified, can tolerate high incubation temperatures.
● Solid media are used in:
○ Slants (test tubes with agar sloped → large surface area).
○ Deeps (agar in tubes for vertical growth, oxygen testing).
○ Petri dishes/plates (flat plates for colonies and experiments).
✅ In short:
● Defined media = exact formula known.
● Complex media = natural extracts, formula not exact.
● Anaerobic media = remove oxygen.
● Selective media = allow some microbes only.
● Differential media = show differences between microbes.
● Special media = for microbes needing unusual conditions.
● Enrichment media = increase small numbers of specific microbes.
