Culture media are the base where microorganisms can grow with the help of nutrients and under the influence of physical growth parameters. All microorganisms cannot grow in a single culture medium and in fact, many cannot grow in any known culture medium. Some organisms that cannot grow in artificial culture medium are known as obligate parasites. Examples: Mycobacterium leprae, Rickettsias, Chlamydias, Treponema pallidum.
Based on the consistent nature of the media, they are
classified into solid, semi-solid, and liquid. Solid media contains agar
(1.5-2.0%) as a solidifying agent and is suitable for the isolation of bacteria
and determination of colony characteristics. Examples: Nutrient agar, BAP, etc.
Semi-solid media also contains agar but the percentage content is less (0.5%).
This media is useful for the determination of the motility of bacteria and the cultivation
of microaerophilic bacteria. Liquid media that do not contain agar except for the
required amount of nutrients. This media is useful for the propagation of a large
number of organisms, fermentation studies, and various tests (Sugar
fermentation test).
There are various types of culture media based on the
applications as well as media compositions.
Flowchart: Various types of culture media
The importance of culture medium is to grow microorganisms
outside the body for some important applications such as:
• To identify the cause of infection from the clinical
samples.
• To study the characteristics and properties of
microorganisms.
• To prepare biological products like vaccines, antigens,
toxoides, etc.
Composition of Culture Medium:
• Energy source
• Carbon source
• Nitrogen source
• Water
• Specific growth factors
• Mineral salts
Synthetic medium: This medium is prepared from
purified ingredients so that the whole composition of the medium is known. They
are simple or complex, based on the supplements incorporated in the medium. A
simple non-synthetic medium has all the nutrient requirements of organisms and
some growth factors whereas complex non-synthetic medium does not contain
purified or completely characterized or constant consistency between their
batches and this medium supports the growth of more fastidious microorganisms
(has a complex nutritional requirement). Example: Nutrient broth is
derived from cultures of yeasts.
1. General purpose media/Basic media: Basal media are
simple media that support most non-fastidious bacteria and are used for the
isolation of primary microorganisms. Peptone water, nutrient broth, and
nutrient agar are considered as basal medium.
2. Enriched medium: Addition of extra nutrients in
the basal medium in the form of blood, serum, egg yolk, etc. to make the medium
enriched. They are used to grow nutritionally exacting bacteria. Examples:
Blood agar, chocolate agar, etc.
3. Selective and enrichment media: They are designed
to inhibit unwanted contaminating bacteria and help to recover pathogens from a
mixture of bacteria. Selective media are agar-based and enrichment media are
liquid in consistency. The addition of antibiotics, dyes, chemicals, alteration
of pH, etc. Makes a medium selective.
They are of various types like:
1. Thayer Martin Agar: It is used to recover N. gonorrhoeae;
contains antibiotics; vancomycin, colistin, and nystatin.
2. Mannitol salt agar and salt milk agar: They are used to
recover S. aureus; and contain 10% NaCl.
3. Potassium tellurite: This medium is used to recover C.
diphtheriae; contains 0.04% potassium tellurite.
4. Mac Conkey’s Agar: It is used for Enterobacteriaceae
members and contains bile salt that inhibits commonly gram-positive bacteria.
5. Pseudosel Agar or Cetrimide Agar: It is used to recover
P. aeruginosa contains cetrimide (antiseptic agent).
6. Crystal Violet Blood Agar: It is used to recover S.
pyogenes and contains 0.0002% crystal violet.
7. Lowenstein Jensen: This medium is used to recover M.
tuberculosis and is made selective by incorporating malachite green.
8. Wilson and Blair’s Agar: It is used for recovering S.
typhi and is rendered selective by the addition of the dye brilliant green.
Enrichment Culture Medium:
Enrichment medium is liquid media, used to increase the
relative concentration of certain microorganisms in the culture prior to
plating on a solid selective medium. It is mainly used as a broth medium.
Example: Selenite F broth, tetrathionate broth, and alkaline peptone water are
used to recover pathogens from fecal specimens.
Differential/Indicator Medium:
In this medium dyes, metabolic substrates, etc. are added so
that the bacteria that utilize them appear as differently colored colonies.
Such media are called differential media or indicator media. Differential media
allow the growth of more than one microorganism but with morphologically distinguishable
colonies.
Examples:
1. Mannitol salts agar (mannitol fermentation = Gram-positive
bacteria Staphylococcus aureus produces yellow colonies with yellow zones.
2. Blood agar (various kinds of hemolysis i.e. α, β, and γ
hemolysis).
3. Mac Conkey Agar (lactose fermenters, pink colonies
whereas non-lactose fermenter produces pale or colourless colonies.
4. TCBS (Vibrio cholerae produces yellow colonies due to
fermentation of sucrose).
Transport media:
These media are used when a specimen cannot be cultured soon
after collection. This media prevents drying (desiccation) of the specimen,
maintains the pathogen-to-commensal ratio, and inhibits the overgrowth of
unwanted bacteria. Examples: Cary Blair's medium, Amie’s medium, Stuart’s
medium, etc. Stuart’s and Amie’s media are semi-solid media in consistency. The
addition of charcoal serves to neutralize inhibitory factors. Cary Blair
transport medium is used to transport feces from suspected cholera patients.
Pike’s medium is used to transport streptococci from throat specimens.
Anaerobic media:
Anaerobic media is mainly supplemented with hemin and
vitamin K where bacteria need low oxygen content, reduced oxidation-reduction
potential, and extra nutrients for their growth. The media is also contains 1%
glucose, 0.1% thioglycollate, 0.1% ascorbic acid, 0.05% cysteine, or red hot
iron filings. Before use the medium is to be boiled in a water bath to expel
any dissolved oxygen and then sealed with sterile liquid paraffin. Example:
Robertson Cooked Meat (RCM) medium is commonly used to grow Clostridium spps. Contains
a 2.5 cm column of bullock heart meat and 15 ml of nutrient broth.
Thioglycollate broth contains sodium thioglycollate, glucose, cystine, yeast
extract, and casein hydrolysate.
Assay Media:
These media are used for the assay of amino acids, vitamins,
and other antibiotics. Antibiotic assay media are used for the determination of
antibiotic potency (by microbial assay).
Raw Materials and Nutritional Requirements for Culture Media
Raw materials play an important role in media preparation. The quality of media depends on the quality of raw materials. The most important raw materials used for preparations of media are water, agar, peptone, casein hydrolysate, meat extract, yeast extract, and malt extract.
1. Water as solvent: Water plays a vital role in the preparation
of media. Water should be free from copper ions because copper ion inhibits the
growth of microorganisms. The conductivity of water should be less than 15 µS
(microsiemens) and the pH of the water should be slightly acidic but should not
be less than 5.5.
2. Petri dishes: Petri dishes are generally
sterilized with ethylene oxide (EtO) or gamma irradiated. If EtO is sterilized
they should be then checked for residual EtO toxicity which affects the growth
of the microorganisms. The maximum permissible limit for residual EtO is 1
µg/g. Only borosilicate glassware should be used because soda glass can leach
alkali into the media.
3. Energy source: The most common substance added to
culture media is glucose which acts as a source of energy and also increases
the rate of growth of organisms. Other carbohydrates are also added to media at
5-10 g/lt as biochemical substrates to detect the production of specific
enzymes in the identification of organisms.
4. Nutrients: The nutrients of culture media are
selected to recover the required spectrum of organisms in the sample e.g.
coliforms or anaerobes. Proper nutrients help with bacterial growth.
5. Essential Metals and Minerals: The inorganic
essential components of culture media are divided on a semi-quantitative basis:
Macro-components (gm/liter): Na, K, Cl, P, S, Ca, Mg, Fe. Micro-components
(mgm-microgm/liter): Zn, Mn, Br, B, Cu, Co, Mo, V, Sr, etc.
6. Buffering Agents: The pH of a culture medium is
poised around the optimum necessary for the growth of the desired
micro-organisms. The use of buffer compounds at specific pK values is
especially necessary when fermentable carbohydrates are added as energy
sources. Such examples of buffering agents are phosphates, acetates, citrates,
zwitterion compounds, and specific amino acids that are added to the culture
media.
7. Indicator Substances: The addition of colored
indicator substances in the medium is very effective in detecting the fermentation
of specific carbohydrates. They change color distinctly and rapidly at critical
pH values. Examples: phenol red, bromocresol purple, fuchsin, etc., are toxic
and it is essential to use in low concentrations. Known sensitive strains of
micro-organisms are also used in the screening tests.
8. Selective Agents: Chemicals or antimicrobials are
added to culture media to make them selective for certain micro-organisms. They
are added at specific concentrations to suppress the growth of unwanted
organisms in a polymicrobial sample. Examples: bile salts, dye-stuffs,
selenite, tetrathionate, tellurite, and azide. Thereafter antimicrobial agents
are used in mixtures when suppressing polymicrobial contaminating flora.
Antimicrobials are more specific in their selective action.
9. Gelling Agents: Gelatin is still used for a few
specific media and carrageenans, alginates, silica gel, and polyacrylamides are
sometimes used as gelling agents but the most important gel-forming substance
used in culture media is agar. It is inert to microbial action and melting
temperatures (38°C and 84°C respectively) the high gel strength allows low
concentrations of agar to be used. It has low toxicity and is also not
hydrolyzed by agarase enzymes in high temperatures. Microbiological agar is
specially processed to yield low toxicity, high clarity, low mineral, and high
diffusion gel.
10. Other Components: Many other substances are added
to culture media for specific purposes like growth factors for fastidious
organisms, pH-reducing compounds for anaerobic organisms (thioglycollate and
cysteine), whole blood to detect hemolytic enzymes, etc.
11. Meat extract: It is obtained by hot water
extraction of lean beef and then concentrated by evaporation. It contains
gelatin, albumoses, peptones, proteoses, amino acids, creatinine, purines, and
accessory growth factors.
12. Yeast extract: It is prepared from washed cells
of bakers’ yeast and contains a wide range of amino acids, growth factors, and
inorganic salts.
13. Malt extract: It is prepared by extracting
soluble materials from sprouted barley in water at 55oC and concentrated by
evaporation. It contains maltose, starch, dextrin, glucose, and small amounts
of protein and protein breakdown products and growth factors.
Nutrition Required for Bacterial Culture Medium
Most of the microorganisms are grown in specific culture
media in laboratory conditions and for proper growth, nutrition plays an
important role in the structural and functional properties of the cells.
Nutrition is generally composed of:
• Major macronutrients: C, H, O, N, S, P, K, Mg, Fe, Ca and
Mn.
• Major micronutrients: Zn, Co, Cu, and Mo.
• Carbon and energy sources.
• Growth factors.
• Vitamins.
Major Macronutrients:
• Carbon: The main source is organic compounds and CO2.
It is the main component of cellular materials.
• Hydrogen: The main source is organic compounds. It
is the main component of cell water.
• Oxygen: The main source is organic compounds, CO2
and O2. It is the main constituent of cell material and cell water.
It is an electron acceptor in aerobic respiration.
• Nitrogen: The main source is organic compounds, NH3,
NO3, N2 etc. It is the main constituent of amino acids,
nucleic acids nucleotides, and coenzymes.
• Sulfur: The main source is organic sulfur
compounds. It is the main constituent of some amino acids like cysteine,
methionine, glutathione, and several coenzymes.
• Phosphorus: The main source is inorganic
phosphates. It is the main component of nucleic acids, nucleotides,
phospholipids, etc.
• Potassium: The main source is potassium salt. It is
the main component of cellular inorganic cation and cofactor for certain
enzymes.
• Magnesium: The main source is magnesium salt. It is
the main component of inorganic cellular cation, a cofactor for certain
enzymatic reactions.
• Iron: The main source is iron salt. It is the main
component of cytochromes and certain non-heme iron proteins and a cofactor for
some enzymatic reactions.
• Calcium: The main source is calcium salt. It is the
main component of inorganic cellular cation, a cofactor for certain enzymes,
and a component of endospores.
• Manganese: The main source is manganese salt. It is
the main component of inorganic cellular cation, a cofactor for certain
enzymes.
Major Micronutrients:
They are also known as trace elements because they require
very small amounts. They usually act as cofactors for essential enzymatic
reactions in the cell. They are cations required for bacterial nutrition.
Examples: Mn, Co, Zn, Cu, and Mo.
Zn2+ is present at the active site of some
enzymes but is also involved in the association of regulatory and catalytic
subunits in E. coli aspartate carbamoyl transferase. Mn2+ aids many
enzymes in catalyzing the transfer of phosphate groups. Mo2+ is
required for nitrogen fixation and Co2+ is a component of Vitamin B12.
Cu2+ is an important micronutrient required as a redox co-factor of
multiple proteins.
Carbon and Energy Sources for Bacterial Growth:
A source of carbon as an energy source and other nutrients
as growth parameters are required in a permissive range of physical conditions
viz. O2 concentration, temperature, and pH. Sometimes bacteria are
referred to as individuals or groups based on their patterns of growth under
various chemical or physical conditions. For example, phototrophs are organisms
that use light as an energy source; anaerobes are organisms that grow without
oxygen; thermophiles are organisms that grow at high temperatures.
All living organisms require an energy source. Organisms
that use light energy are called phototrophs, while organisms that use an
organic form of carbon are called heterotrophs, and when organisms that oxidize
inorganic compounds are called lithotrophs. The source of carbon is either
organic carbon or CO2. Organisms that use organic carbon are
heterotrophs and organisms that use CO2 as a sole source of carbon
are known as autotrophs (Table).
Table: Prokaryotes as per nutritional requirement
|
Nutritional Type |
Energy Source |
Carbon Source |
Examples |
|
Photoautotrophs |
Light |
CO2 |
Cyanobacteria,
some Purple and Green Bacteria |
|
Photoheterotrophs |
Light |
Organic
compounds |
Some Purple
and Green Bacteria |
|
Chemoautotrophs
or Lithotrophs |
Inorganic
compounds, e.g. H2, NH3, NO2, H2S |
CO2 |
A few
Bacteria and many Archaea |
|
Chemoheterotrophs
or Heterotrophs |
Organic
compounds |
Organic
compounds |
Most
Bacteria, some Archaea |