Culture media are the artificial media that are
designed to grown microorganisms in the form of liquid, semi-liquid or solid media.
Importance: Culture media
helps to isolate one particular microorganism group from a mixture. It provides
reliable ways to estimate number of microorganisms. It helps to manipulate
microorganisms to some genetic studies.
Microorganisms in the environments depend on various factors such as
nutrients, oxygen, temperature and moisture for their survival, growth and
reproduce. When Chemoheterophic microorganisms are
grown in artificial medium in the laboratory, it has to be supplied externally
carbon source, nitrogen source and energy source. Chemoheterophic
microorganisms refer to as oxidize organic or inorganic substrate to produce
ATP and start their carbon nutrition form organic compounds which are produced
from carbon dioxide by autotrophs.
Culture media must provide basic
requirements for microorganisms those are needed to grow. Agar is added as the solidifying agent.
1.0-1.5% agar is added to prepare solid culture media. Semi-sold culture media are
prepared by adding 0.5%agar. Carbon
source is added as the oxidizable substrate for microorganisms to
synthesize their ATP. Lactose is usually added as carbon source. Nitrogen and other nutrients are
provided as beef extract. Usually peptone is added to provide both carbon and
nitrogen with other nutrients to the medium. Peptone is a mixture of partially
digested protein of meet intrusion. It contains proteins that are readily
digestible for microorganisms. It fastidious microorganisms which has
complex nutrients requirement, it has to be supplied special nutrients. Serum
or yeast extract or beef extract is added to fulfill their special nutrient
requirements. It selective culture media is needed to prepare to isolate one
microorganism group, antibiotics is added to kill other microorganisms. In
addition to those sources, electron donors and acceptors and growth promoting
vitamins have to be added.
Consistency of the medium is modified
by adding solidifying agent such as agar,
gelatin or albumin. Earlier gelatin was used as solidifying agent. But
gelatin had some disadvantages. It is liquid at 37◦ C. It is digested by many
bacteria. Therefore later agar which is extracted from red algae is used as solidifying
agent. Solid agar melts at 85◦ C. it is not preferably digested by bacteria.
In
addition above nutrients requirements, chemical and physical conditions also
must be adjusted for growth of microorganisms.
Majority of microorganisms are able to survive at neutral pH. Few prefer
extremely acidic or basic pH. Oxygen is also major limiting factor that affect
the growth of microorganisms. Aerobic, facultative and microaerophilc are able
to survive and grow in the presence of oxygen. But anaerobic microorganisms cannot survive in the
presence of oxygen. Therefore if anaerobic microorganisms are needed to grow,
culture media must be free of oxygen.
Addition of reducing substance, displacement oxygen by carbon dioxide
and boiling are the methods that help to create oxygen free environment.
Microorganisms show maximum growth at their optimum temperature. According to
the temperature requirements, microorganisms are categorized into three groups
such as psychrophilic, mesospheric and thermophile. Psychrophilic grow and
reproduce at 0◦ C. Mesospheric grow and reproduce at 15-30◦ C. Thermophilc
grown even greater than above 90◦ C. Moisture content also effects the growth
of microorganisms.
Nutrient agar (NA) is the
general purpose media for bacteria in the laboratory. It is prepared by adding
beef extract (3g), peptone (5g), agar (15g) and distils water (1000ml). The pH
of the culture media is 6.8±0.2. Bacteria in the free environment are
inoculated at 25◦ C for 48 hours. But human pathogens are inoculated at 37◦ C
because of inner body temperature of human.
Some bacteria show growth even after 24 hours because growth rate
depends on the species.
Dextrose potato agar (DPA) is the
general purpose media for fungi in the laboratory. It is prepared by adding dextrose
(20g), potato extract (4g) and agar (15g). Fungi are inoculated at 25◦ C for
5days. Fungi have relatively lower growth rate compare to the bacteria. The pH
of the culture media is 6.2.
Culture
media are classified according to their consistency, usage, purpose and
nutrient composition.
According
to consistency, culture media are
divided into three categories such as liquid,
semi- solid and solid. Broth
culture is the example for liquid media. It helps to observe motility and
characteristics of bacteria. Semi- solid media are prepared by adding 0.5%agar.
It gives the advantage of observing motility of bacteria. It provides ideal
environment for inoculating microaerophilic bacteria. Solid media are prepared
by adding 1.0-1.5% agar. Slop media (slunt) and deep stab are the example for
solid media. In slunt, slope provides the high surface area for inoculation.
Slunt is used to inoculate aerobic bacteria while butt is used to inoculate
anaerobic bacteria. Deep stab is used to observe the growth of bacteria
according to oxygen availability. Top area of deep stab is used to inoculate
aerobic bacteria while bottom of the deep stab is used to inoculate anaerobic
bacteria.
Culture
media are categorized into two groups such as general purpose and special purpose media based on the purpose. General purpose media is
comprised with basic nutrients requirements. These media contains only carbon
source and nitrogen source .Special purpose media supports the growth of
fastidious microorganisms. So in addition to carbon source and nitrogen source,
those media contain special nutrients.
Culture
media are categorized into simple,
complex and defined (or synthesis) according to the nutrient composition. Simple
media supports the non-fastidious microorganisms. It includes peptone, water
and nutrient agar. Complex media supports the fastidious microorganisms. Exact
chemical composition of complex media is unable to find. Blood agar is one of
the examples for complex media. Defined (or synthesis) media is used for
researches. Exact chemical composition of the defined media is known.
Culture
media are classified into basal, enrich, selective or isolation, differentiate,
reducing and transport based on their usage.
Basal media support the growth of non-fastidious microorganisms. It is also
made of peptone, nutrient broth or nutrient agar. Enriched media are used to grow fastidious microorganisms. Their special
nutrient requirement is fulfilled by addition of blood serum, egg york into
basal media. Differential/Indicator
media helps to isolate one microorganisms form other microorganisms. Their
metabolic activates help to isolation of them. As an example, neutral red or bromo
cresol purple is added as the indicator to detect the acid production in
coliform test. Coliform are the special group of bacteria that are able to
produce acids during their sugar fermentation process. Selective and enrichment media are used to inhibit the growth of
untargeted or unwanted microorganisms in a mixture. Addition of antibiotics and
alteration of pH of the media help to inhibit of the untargeted or unwanted
microorganisms. As an example, Brilliant Green Lactose Bile (BALB) broth
contain bile to suppress the growth of gram positive bacteria in the confirm
test of fecal coliform. Transport media
are useful for bringing clinical sample into the laboratory. Those media
supports to inhibit the overgrowth of unwanted microorganisms in a pathogen mixture. Saline is added to prevent the desiccation.
Charcoal is added to neutralize the inhibitory factors. Reducing or anaerobic media are used for inoculation of anaerobic
microorganisms. Oxygen can be removed by using either physical or chemical
methods. As a psychical method, boiling removes oxygen from the media. Red hot
iron filling can be added to remove oxygen as a chemical method.
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