CULTURING

Definition of Terms

1. Medium: The substance which provides nutrients for the growth of microorganisms
2. Culture medium: The nutrients on which microorganisms are cultivated
3. Microbial Culture: A process of growing and multiplying microorganisms in a laboratory
setting under controlled conditions
4. Inoculation: The process of introducing a small amount of microorganisms (inoculum) into a
culture medium to initiate their growth.
5. Inoculum: The small amount of microorganisms used to initiate a culture. It can be a liquid
suspension or a solid sample containing microorganisms.

Types of Culture Media

 One of the most important reasons for culturing bacteria in vitro is its utility in diagnosing
infectious diseases. Isolating a bacterium from sites in body normally known to be sterile is
an indication of its role in the disease process.
 Culturing bacteria is also the initial step in studying its morphology and its identification.
Bacteria have to be cultured in order to obtain antigens from developing serological assays or
vaccines.
 Certain genetic studies and manipulations of the cells also need that bacteria be cultured in
vitro. Culturing bacteria also provide a reliable way estimating their numbers (viable count).
Culturing on solid media is another convenient way of separating bacteria in mixtures.
Importance of microbial culturing

1. Identification of Microorganisms: Culturing microorganisms allows for the identification and

classification of different species and strains. By observing their growth characteristics,
colony morphology, and conducting biochemical tests, scientists can determine the type of
microorganism present in a sample.
2. Study of Microbial Physiology and Metabolism: Culturing microorganisms provides a
controlled environment for studying their physiology, metabolism, and growth requirements.
3. Development of Diagnostic Tests: Microbial culturing is essential for developing diagnostic
tests to detect and identify pathogenic microorganisms.
4. Drug Discovery and Development: Culturing microorganisms is instrumental in the
discovery and development of new drugs and antibiotics. Many antibiotics are derived from
naturally occurring compounds produced by microorganisms.
5. Environmental Studies: Microbial culturing enables researchers to study the role of
microorganisms in the environment. By isolating and characterizing microbial communities
from soil, water, or other ecological niches, scientists can assess their ecological functions,
nutrient cycling, and interactions with other organisms. This knowledge helps in
environmental monitoring, bioremediation efforts, and understanding the impact of
microorganisms on ecosystem health.
6. Industrial Applications: Microbial culturing is widely used in various industries, including
food and beverage, pharmaceuticals, agriculture, and biotechnology. It allows for the
production of enzymes, biofuels, antibiotics, and other valuable compounds using microbial
fermentation processes. By optimizing culturing conditions and selecting high-yielding
strains, industries can achieve efficient and cost-effective production of desired products.

Composition of Culture Media:

Bacteria infecting humans (commensals or pathogens) are chemoorganoheterotrophs. When
culturing bacteria, it is very important to provide similar environmental and nutritional
conditions that exist in its natural habitat.Hence, an artificial culture medium must provide all the
nutritional components that a bacterium gets in its natural habitat. Most often, a culture medium
contains water, a source of carbon & energy, source of nitrogen, trace elements and some
growthfactors. Besides these, the pH of the medium must be set accordingly. Some of the
ingredients of culture media include water, agar, peptone, casein hydrolysate, meat extract, yeast
extract and malt extract.

Classification of Culture Media

Bacterial culture media can be classified in at least three ways;

1. Based on consistency
2. Based on nutritional component
3. Based on its functional use

Based on Consistency

Culture media are liquid, semi-solid or solid and biphasic

Liquid media:

 These media do not contain any traces of solidifying agents. They are available for use in
test-tubes, bottles or flasks. Liquid media are sometimes referred as “broths” (e.g nutrient
broth). In liquid medium, bacteria grow uniformly producing general turbidity.
 Certain aerobic bacteria and those containing fimbriae (Vibrio & Bacillus) are known to
grow as a thin film called ‘surface pellicle’ on the surface of undisturbed broth. Bacillus
anthracis is known to produce stalactite growth on ghee containing broth. Sometimes the
initial turbidity may be followed by clearing due to autolysis, which is seenin penumococci.
 Long chains of Streptococci when grown in liquid media tend to entangle and settle to the
bottom forming granular deposits.
 Liquid media tend to be used when a large number of bacteria have to be grown. These are
suitable to grow bacteria when the numbers in the inoculum is suspected to be low.
Inoculating in the liquid medium also helps to dilute any inhibitors of bacterial growth.
 This is the practical approach in blood cultures. Culturing in liquid medium can be used to
obtain viable count (dilution methods). Properties of bacteria are not visible in liquid media
and presence of more than one type of bacteria cannot be detected.
Advantages of liquid media

1. Identification of mixed cultures growing in liquid media requires subculture onto solid media
so that isolated colonies can be processed separately for identification.
2. Growth in liquid media also cannot ordinarily be quantitated.
3. Bacteria grown in liquid cultures often form colloidal suspensions

Composition and Uses of Some Common Liquid Media

Preparation of a Selected Liquid Media

1. Pepetone Water

Meaning: It is a broth medium used for the growth of the organism and a base for
determining carbohydrate fermentation patterns of non-fastidious organisms. In addition, it is
also used for the detection of indole production by the organism

Composition:

Ingredients Gms / L

Peptone 10.0

Sodium chloride 5.0

Principle of Peptone Water

The two basic components of peptone water are peptone and sodium chloride. Peptone provides
nitrogenous and carbonaceous compounds, long-chain amino acids, vitamins provide essential
nutrients. Sodium chloride provides the necessary electrolyte and maintains the osmotic balance
of the medium.

Preparation of Peptone Water

1. Suspend 15.0 grams in 1000 ml distilled water.

2. Mix thoroughly and distribute into the final containers.

3. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes.

Uses of Peptone Water

1. Peptone Water is used as a growth medium and as a base for carbohydrate fermentation
media.

2. It is a broth medium used for the detection of indole.

3. It is minimal growth medium which is used for determining carbohydrate fermentation

patterns of non-fastidious organisms.

4. It is useful as a diluent or for making suspensions of non-fastidious microorganisms for

microbial enumeration procedures.

5. Peptone Water with pH adjusted to 8.4 (alkaline condition) is suitable for the cultivation and
enrichment of Vibrio species.

2. Nutrient Broth

Meaning: Nutrient Agar is a basic culture medium commonly used for the culture of non-
fastidious microorganisms, and for quality control and checking purity prior to biochemical or
serological testing.

 Nutrient media can also be used for the cultivation of fastidious microorganism by enriching
the medium with serum or blood

 Nutrient agar is a general-purpose media that is mostly used for routine culture or to ensure
prolonged survival of microorganisms.

 It is one of the most important and commonly used non-selective media for the routine
cultivation of microorganisms.

 Nutrient agar has been used for the cultivation and enumeration of many bacteria that are not
particularly fastidious.

 The media can be made suitable for the cultivation of other fastidious organisms by the
addition of different biological fluids such as horse or sheep blood, serum, egg yolk, etc.
Principle of Nutrient Agar

 Nutrient agar is made with various nutrients which allow the growth of a wide variety of
microorganisms that do not usually require specific nutrients or supplements.

 The primary constituents of the media are peptone, beef extract, and agar. In addition to these
nutrients, some vitamins and some trace ingredients necessary for the growth of bacteria are
also added.

 The peptone is the source of nitrogen or protein that acts as a source of amino acids for the
bacteria.

 The beef extract is the primary source of carbon which is essential for the formation of
carbohydrates in the bacteria. It also contains other components like some vitamins, different
trace minerals, organic compounds, and salts, which further enhance the growth of different
organisms.

 Besides, sodium chloride is added to the medium in order to maintain the osmotic
equilibrium of the medium and prevent the change in pH of the medium during growth.

 The distilled water provides a medium to dissolve the nutrients so that it is easier for the
bacteria to absorb them.

 Agar is the solidifying agent that provides a stable surface for the organism to grow on,
which allows for the observation of colony morphology and enumeration of the organism.

 The medium is made up of basic nutrient that is essential for a wide variety of organism,
which makes it a general media that can be used for various purposes.

Composition of Nutrient Agar

 Most nutrient agar used these days in laboratories is prepared from the dehydrated powder
supplied by different vendors. The composition of the media, however, remains the same.

 It can also be prepared in the lab if the necessary constituents of the media are available.

 The following is the composition of the nutrient agar:

 S.N Ingredients Gram/liter

1. Peptone 5.0

2. Yeast extract 1.5

3. Beef extract 1.5

4. Sodium chloride 5.0

5. Agar 15.0

Preparation of Nutrient agar

1. In a beaker, 28 grams of the dehydrated powder or lab-prepared media is added to 1000

milliliters of distilled or deionized water.

2. The suspension is then heated to boiling to dissolve the medium completely.

3. The dissolved medium is then autoclaved at 15 lbs pressure (121°C) for 15 minutes.

4. Once the autoclaving process is complete, the beaker is taken out and cooled to a temperature
of about 40-45°C.

5. If enrichment is desired, the addition of blood or biological fluids can be done after the
autoclaving process.

6. The media is then poured into sterile Petri plates under sterile conditions.

7. Once the media solidifies, the plates can be placed in the hot air oven at a lower heat setting
for a few minutes to remove any moisture present on the plates before use.
Storage of Nutrient agar

 The media in the powder form should be stored between 10 to 30°C in a tightly closed
container, and the prepared medium should be stored at 20-30°C.

 After opening, the product should be appropriately stored when dry, after tightly capping the
bottle in order to prevent lump formation as the medium is hygroscopic in nature and thus,
absorbs moisture relatively quickly.

 The container should be stored in a dry ventilated area protected from extremes of
temperature and sources of ignition.

 The product should be used before the expiry date on the label.

Uses of Nutrient agar

 Nutrient agar is used for the culture of less fastidious organisms as a general medium.

 It is also used for routine culture of microorganisms from typical environmental samples like
water, food, and even air.

 Nutrient agar is often used for the demonstration and teaching purposes as it doesn’t contain
harmful substances and can be used for the isolation of multiple microorganisms.

 The use of Nutrient agar is recommended by standard methods as it has a simple composition
which can even be prepared within a laboratory.

 It can also be used for the preservation of microorganisms for an extended period of time
without contamination that might occur in a more nutritious medium.

 It is used as a purity testing method prior to the different biochemical tests and serological
tests.

 It is one of the most common media used for the enumeration of bacteria from environmental
samples.

 The addition of biological fluids like horse or sheep blood, serum, egg yolk, etc. to the
nutrient agar makes the medium more selective for certain fastidious organisms.
Solid Medium

If agar is added to a nutrient broth, it becomes solid medium. It is used for isolating microbes and
to determine characteristics of colonies. By varying the concentration of agar, it is possible to
make the medium solid or semisolid. It remains solid on incubation and not destroyed by
proteolytic bacteria.

Composition and Uses of Some Common Solid Media