Section II
Culturing Techniq.ue
Experiment Number 1. Preparation of Culture Media
All living beings includihg microorganisms require basic nutrients for living. For any bacterium to be
propagated for any purpose such as to characterize the morphological, physiological and biochemical
properties of microbes, laboratory cultivation of microbial cell is necessary. For the cultivation of bacteria
in laboratory, it is necessary to provide the appropriate biochemical and biophysical environment. The
biochemical i.e. nutritional environment is made available as a culture media.
A bacteriological culture medium is the artificial nutritional preparation for the cultivation of bacteria in
the laboratory. Simply, the materials upon which we grow bacteria in the laboratory are called culture
media. In constructing a culture medium for any microorganisms, the primary goal is to provide a balanced
mixture of the required nutrients (food, energy and growth factors) at concentration that will permit the
rapid microbial growth. The medium also provides the necessary moisture and controls the pH of the
environment.
Robert Koch in 1843-1910 discovered and developed the use of culture media for the cultivation of
bacteria. He introduced potato agar as the first medium in bacteriology. For the growth of particular
organisms, it is necessary to know their nutritional requirements and physical conditions. It is important
to note that no one medium will support the growth of all microorganisms . Due to the wide range of
nutritional requirements of microorganisms, large variety and types of culture media have been developed
with differen1purpose and uses .
Culture media are employed by the bacteriologists for the isolation , growth and maintenance of pure
culture of bacteria and also used for identification of bacteria according to their biochemical and physiological
properties.
Common Ingredients of culture Media
The following are the common ingredients, which are found is many culture me.dia .
Agar: In solid media, Agar or Agar-Agar is the major solidifying agent and is now universally used for
preparing solid media.
It is an impure polysaccharide obtained from certain red marine algae belonging to the class
Rhodophyceae. It consists for two main polysaccharides, agarose (70- 75% ) and agaropectin (20-25% ).
Agar is a complex polysaccharide consisting of 3, 6 anhydro-L-galactose and 0-galacto-pyranose , free of
nitrogen.
Agar has no nutrient value to the bacteria and cannot be hydrolyzed by most commonly cultured
bacteria. The significance of this is that it can he used as a solidifying agent for bacterial media without
changing the components of the medium or without liquefying during the time the bacteria are growing.
Agar has several other properties that are also of great importance to the microbiologist.
24 Practical Approach to Microbiologij
Agar is a reversible colloid that can change from the liquefied phase to the gel (solidified) phase at
certain temperatures. The agar liquefies at about 100°C but the reverse change from liquefied to solidified
state occurs at about 45"C. Solidified agar is slightly cloudy, but still somewhat transparent. This is an
important attribute because it is possible to observe many characteristics of bacterial colonies that would
not be apparent if the solidifying agent were opaque.
Previous to agar, potato slices or gelatin were used to form solid substrates for microbial cultivation .
But, these materials are unacceptable as they are degraded by a wide variety of microorganisms.
Furthermore gelatin liquefies when the temperature is raised above 25°C and potato slices are opaque.
Peptone: Peptone is a water-soluble products obtained from the breakdown i.e. hydrolysis of animal or
plant proteins such as meat scraps, muscles, gelatin, milk protein (casein) and soybean meal. They are
hydrolyzed by acids or enzymes such as pepsin, trypsin and papain. The exact composition of peptone
will depend on the raw material and the method of manufacturer. Peptones primarily contain peptides,
single amino acids, and proteases .
In media, peptone provides nitrogen for growing microorganisms. It should be light in colour, dry and
have a neutral pH . The concentration of peptone used depends on the uses of individual culture media.
Three examples of brand names of peptones are tryptone or tripticase (a pancreatic digest of casein),
phytone or soytone (a digest of soybean meal) and simple peptone (a digest of beef muscle). Peptones
are_also used frequently in combination with extracts for the cultivation of fastidious organisms. Simple
peptone solution also supports.the growth of many organisms.
Extracts (Meat extracts and yeast Extracts): Eukaryotic tissues (yeast, beef muscle, liver, brain, heart
etc.) are extracted by boiling and then concentrated to a paste or dried to powder. Those, which are
extracted from yeast cells, are called yeast extract and those from meats are called meat extracts.
These extracts are frequently used as a source of amino acids, vitamins, and mineral salts including
phosphates and sulphates. Besides, trace elements and some sugar are also present. Beef extracts
such as laboratory Lem co provides organisms with a further supply of amino acids, and also with essential
growth vitamins and mineral salts. It is an ingredient of many culture media including nutrient agar and
nutrient broth. Trypsin digested meat extracts are also used in some media. As meat extracts are readily
available and easy to use they have largely superseded fresh meat infusions, which are both time consuming
to prepare and variable in quality.
Yeast extracts a.re made from bakers' or brewers' yeast and is a rich source of amino acids and
vitamins of the B-complex. In culture media it is used to supplement or replace meat extracts. Yeast
extracts are found in XLD, MNYC, and iCBS media etc.
Body Fluids: Whole or defribrinated blood , plasma, serum or other body fluids are frequently added to
culture media for the isolation and cultivation of many pathogens . Body fluids contribute many growth
!actors and substances that detoxify certain inhibiters .
Blood- The choice of blood is often a matter of convenience and depends on the animals kept by a
aboratory. When bought from a commercial source, commonly used is the horse or sheep blood, but the
'.:}lood of other species may be used for special purposes . Blood must be stored in a refrigerator but
should not be allowed to freeze .
Plasma- Plasma is used for demonstrating coagulase activity. In medical microbiology human plasma
s preferred but rabbit plasma may be used . Plasma may be obtained by removing the supernatant of
Practical Approacl1 to Microbiology 25
blood and anticoagulant in which the red cells have settled. Liquid plasma is an unstable product, liable
to coagulate or to form particles that cause a turbidity or deposit. It should not be filtered. Plasma should
be stored in a refrigerator but should not be frozen.
Serum- Serum is prepared from blood, collected without addition of an anticoagulant, by removal of
the liquid which separates when the clot is separated. Serum should be sterilized by filtration .
Bile and bile salts- Bile contains several bile acids as compounds conjugated with amino acids. Bile
acids can also form addition compounds with higher fatty acids and other substances. Bile also contains
the pigments, billirubin and billiverdin. Fresh ox bile has been superseded either by bile extracts, dehydrated
bile or bile salts. Commercial bile salts are prepared by extracting dried ox or pig bile with ethanol,
decolorizing the extract with charcoal, and precipitating the bile salts with ether to form a water-soluble
yellowish brown hygroscopic powder. When prepared from ox bile, the salt consists mainly of sodium
taurocholate and sodium glycocholate with smaller amounts of the sodium salts of taurodeoxycholic and
glycodeoxycholic acids.
Carbohydrates: The bacteriologists use carbohydrates (collectively called 's.ugars') to enrich media for
growth, pigmentation, and to determine whether the organism can produce acid or acid and gas from
them. The concentration of carbohydrate used in oxidation of fermentation studies is 0.5-1%. Some
carbohydrate solutions may be sterilized by autoclaving whereas with others decomposition may occur.
The most commonly used carbohydrates are glucose (dextrose), sucrose, lactose, mannose, galactose,
fructose, cellobiose, maltose, starch etc. These simple and complex sugars are added to many culture
media to provide bacteria with source of carbon and energy.
Mineral salts: Sulphates are required as source of sulphur and phosphates as source of phosphorous,
which are very essential for microbial cell growth. Culture media should also contain traces of Mg, K, Fe,
Ca and other elements, which are required for bacterial enzyme activity. NaCl is also an essential ingredient
of most culture media. NaCl is added to adjust the osmotic pressure of the medium so that it is isotonic
for the bacterial cell.
Buffers: Buffers are incorporated to maintain the optimum pH of the media. During microbial culture they
produce acidic or alkaline products, which may alter the pH of media. This alteration is pH can be •
controlled by adding buffer. Substances like sodium and potassium phosphates and calcium carbonates
prevent marked changes in pH. Crude organic preparation such as peptone also acts as buffers.
pH Indicators: pH indicators are incorporated in some culture media often in differential media and give
usual evidence of pH changes occurring during the growth of bacteria. Indicators for this purpose must be
non-toxic in the concentration used. There are different types of indicators available for use in media.
Water: Water is essential for the growth of all microorganisms . Water used in microbiology laboratory
should be free from any chemicals that inhibit bacterial growth. Distilled water must be used in the .
preparation of culture media, reagents and for staining purpose if the local water supply has a high mineral :
content. The use of distilled water helps to assure the uniformity of different batches of media. Its use also
prevents the formation of undesirable precipitates in the media during sterilization. Since microbial growth
is determined by the appearance of turbidity, an optically clear nutrient broth is desirable.
Storage of Media: Freshly prepared medium is desirable although with some {e.g. Cary & Blair's bismuth
sulphite agar) maturation is necessary. Many media can be safely kept at room temperature or in a
refrigerator for several weeks or even months, before use. Stability varies with the individual medium and
it is not possible to fix a useful storage life. The important point is that moisture should be retained. To
prevent evaporation and concentration of the constituents when media are to be stored, they should be
kept in screw-capped rather than in cotton-plugged containers. Appreciable evaporation can occur in a
refrigerator but putting closed media containers in polythene bags can prevent this. Other important
points that should be considered during storage of prepared media can be listed as,
If nutrient agar slopes appear to be dry, they should be melted and re-soldified.
Strong light is detrimental to most media and storage in the dark is preferable, especially for those
containing dyes or indicators.
Cotton plugged tubes are a potential source of contamination. The moisture absorbed by the
cotton is sufficient to permit development of fungi, and hyphae may penetrate a cotton plug.
Media, which have been kept in a refrigerator, should be allowed to attain room temperature before
use. Because the solubility of gases in liquids decreases with increase in temperature, it is
essential to check that gas bubbles do not appear in the Durham's tubes of carbohydrate media
as they warm up from storage to room temperature.
Classification of Culture Media
The first requirement for the scientific study of the bacteria is the development of laboratory methods for
~eir growth and preservation. A culture medium is formulated on the basis of the food, energy, growth
factor, and environmental requirements of the organism to be cultured or on the basis of some specific
function, which the medium is supposed to serve. Culture media can be classified according to their
cilemical composition, use and physical state as follows:
A. Classification on the basis of physical state
On the basis of physical state of the media, it can be classified as:
i. Solid Media
Solid media are those media, which are in solid state. Agar is the major solidifying agent used to
prepare solid media. It is added in a powder form at more or less standard concentration ( 1.5%) for
plates and slanted media . Examples of solid media are Nutrient agar, MacConkey agar, Blood
agar etc. The solid media are used for the surface growth of microorganisms, to determine the
colony characteristics of microorganisms and also for the isolation and enu~ation.
ii. Liquid Media
Those media, which are in liquid state, are called liquid media. Liquid media are prepared as the
solid media but the addition of agar is avoided so, the media are in a liquid state.
Examples of liquid media are: Nutrient broth, MacConkey broth, Glucose fermentation broth,
Peptone water etc. Generally, the liquid media are used for the propagation of a large number of
microorganisms, for the transfer of organisms, for sugar fermentation tests, and for some biochemical
tests etc.
iii. Semi-Solid Media
Semi-solid media are those whose consistency is jelly like in nature i.e. semi-solid media are
gelatinous and have consistency in between solid and liquid states. They are prepared by using
either agar or gelatin but in low concentration i.e. 0.1 to 0.5% agar.
Examples of semisolid media are: Hugh and Leifson's media, SIM media, etc. Semisolid media
are useful for the cultivation of microaerophilic bacteria and are often used to detect bacterial
motility.
B. Classification according to chemical composition
On the basis of chemical composition i.e. types of ingredients present in the media; they can be
classified as-
i. Synthetic media (chemically defined} media
ii. Non-synthetic media (complex media}
i. Synthetic media
Synthetic media are generally made from various chemical compounds that are highly purified and
precisely defined. It may contain only inorganic compounds, or both organic and inorganic
compounds.
Synthetic media are prepared from pure chemical substances and the exact composition of the
medium is known both qualitatively and quantitatively. Synthetic media are primarily used in research
work and have the advantage that, wherever prepared, their composition is the same. Therefore the
results of microbial action on these media in one laboratory are strictly comparable with those in
other laboratories thousands of miles away. E.g. of Synthetic media is the following media containing
only inorganic compounds;
Na2Sp3 .5Hp 10 gms/lit
. KH2 P04 4 .0 gms/lit
~HP0 4 4.0 gms/lit
MgSOJHp 0.8 mgs/lit
NH4 CI 0.4 mgs/lit
Trace element solution 10.0ml
Distilled water -----..__
1000 ml.
It is used as an enrichment medium for the isolation of Thiobacil/us thioparus from its natural
habitat.
Synthetic media are of great value in studying the nutritional requirements of microorganisms and
also in studying a great variety of their metabolic activity, mostly the enrichment media comes
under this category.
ii. Non-synthetic Media (Complex media)
Non-synthetic media are such type of media, which contain ingredients of imprecise composition .
Complex media contain all necessary ingredients for growth of microorganisms, but they are in
crude form, that is, not all the components of the media nor their exact quantities are known. It is
often prepared from very complex materials of biological origin such as -blood, milk, yeast, extract,
beef extract, body fluids and peptones etc. the exact chemical composition of which is obviously
undetermined. Complex media usually provide the full range of growth factors that may be required
by an organism such as - polypeptides, amino acids, vitamins and minerals. So they may be
more handily used to cultivate unknown bacteria and bacteria whose nutritional requirements are
complex.
C. Classi.fication according to use
According to use, culture media can be classified into different types for different purpose. They ··
are as follows-
i. General Purpose Media
These are the simple media and are also called basic stock media or basal media. These are
designed to grow as many as possible of the organisms commonly used in general bacteriology
laboratories. But, general-purpose media will not adequately support the growth of microorganisms
with special dietary requirements such as blood or specific amino acids.
They are often used in the preparation of enriched media, to keep the culture in normal condition
or to maintain stock culture, for sub-culturing pathogens from differential or selective media prior to
perform biochemical and serological tests; E.g. Nutrient agar, Nutrient broth etc.
ii. Selective Media
A selective media contains some selective substances that support the growth of desired organisms
while inhibiting the growth of unwanted organisms in a mixture. They facilitate the isolation of
particular species from a mixed inoculum . Commonly used selective agents are bile salts, brilliant
green, sodium dioxycholate, crystal violet, eosin Y, methylene blue, sodium azide etc. Selective
media are used for isolating a particular type of bacteria by adding a selective substance of
desired organisms. For example: XLD agar selects for Salmona/la spp and Shige/la spp by the bile
salts that inhibit the growth of many faecal commensals.
/
MacConkey agar contains selective agents like bile salts and crystal violet which inhibit the
growth of Gram positive bacteria by supporting the growth of Gram negative bacteria.
Media containing sodium azide will support the growth of Gram positive bacteria by inhibiting most
of the Gram negative bacteria.
In recent years . antimicrobials have become increasingly used as selective agents in culture
media; for e.g. , Modified New York City (MNYC) medium for isolating Neisseria gonorrhaeae.
Beside addition of selective agents, one can also adjust the physical conditions of a culture
medium such as pH, and temperature to render it selective for organisms of interest.
iii. Differential Media or Indicator Media
Differential media contain some indicators, dyes or other substances to differentiate microorganisms.
This media is used to differentiate several genera and species of microorganisms having closely
related characteristics . Different microorganisms act in various ways on different components
present in media to produce a variety of end products or effects, which is often detected by
variation in colour of pH indicator.
Examples of Differential media are: Eosin Methyfene Blue (EMB) agar is both selective and differential
medium. It supports the growth of gram negative organisms over that of gram positive organisms
and it will differentiate Escherichia from Enterobacter. Escherichia will produce dark centered
colony with greenish metallic sheen while Enterobacterwill produce a pinkish and viscous growth.
Similarly, MacConkey agar is another example of selective differential media. It differentiates
lactose-fermenting bacteria from lactose non-fermenting bacteria with the help of pH indicator
neutral red.
Example of differential medium that does not involve pH related reactions include Blood agar. It
differentiates haemolytic and non-haemolytic bacteria. Some of the bacteria may destroy red
blood cells and others do not. RBC destroying types of bacteria produce a clear.zone around a
colony and are called haemolytic bacteria, while others, which do not produce a clear zone, are
called non-haemolytic bacteria.
iv. Enrichment Media
In mixed culture or in m~terials containing more than one bacterium, the bacterium to be isolated
is often overgrown by the unwanted bacteria. Usually the non-pathogenic or commensal bacteria
tend to overgrow the pathogenic ones; for e.g., Salmonella typhi being overgrown by E.coli in
culture from faeces.
Similarly in case of cooked food sample, the organisms may be damage.d and may not grow from
direct plating. Hence, in order to get proper isolations, substances, which have stimulating effect
on the growth of desired bacteria and inhibitory effect on those to be suppressed are incorporated
in the liquid media and ar.e called enrichment media . Examples of enrichment media are listed
below.
Tetrathionate broth is an enrichment broth for Salmonella spp . and Shigella spp. It contains
tetrathionate, which inhibits coliform while supporting the growth of Salmonella and Shigella spp.
Similarly; Selenite F broth is an enrichment media only for Salmonella spp .
Alkaline peptone water is used as enrichment medium for Vibrio spp. The pH of media is high
(8.5-9) that supports the growth of Vibrio spp and is inhibitory to other organisms .
v. Enriched Media
Enriched media are used for the cultivation of certain microorganisms that have elaborate
requirements for specific nutrients. Such microorganisms are referred to as fastidious
microorganisms such as Haemophilus influenzae, Streptococcus spp, Neisseria spp. In such
media some additional supplementary foods are supplied beside the common nutrients. Such as
blood, serum, egg, vitamins, extra peptones, amino acids to a basal media. The fastidious
microorganisms growing in enriched media are generally human pathogens. In microbiology
laboratory, these media are used for the diagnostic purpose. Examples of enriched media are
Blood agar, Chocolate agar, Egg yolk medium etc.
vi. Transport Media
Transport media are devised to n:iaintain the viability of a pathogen and to avoid overgrowth of other
contaminants during transit from the patient to the laboratory. These are mostly semi-solid media
containing ingredients to prevent the overgrowth of commensals and ensure the survival of aerobic
and anaerobic pathogens when specimens cannot be cultured soon after collection. Their use is
particularly important when transporting microbiological specimens from health centers to the"
district microbiology laboratory.
Examples of transport media include Cary and Blair medium for preserving enteric pathogens,
Amies transport medium used to prevent the viability of gonococci and other pathogens in specimens
collected on swabs and viral transport medium for viruses etc.
Yii. Anaerobic Culture Media
The early bacteriologists believed that successful cultivation of obligate anaerobes could only be
achieved through methods that excluded all free oxygen. Recently, it has become apparent that
providing adequate anaerobic conditions requires only the removal of free oxygen from the immediate
environment of the microorganisms, or simply the maintenance ofa low oxidation-reduction potential
in media. Quite often, the same media may be used for both aerobes and anaerobes, providing
that these media support adequate growth .
An anaerobic bacterial species is one that cannot grow in the presence of free oxygen . Oxygen
is toxic to these bacteria. In some instances, very small amounts of oxygen are toxic and the
requirement for an oxygen-free environment is absolute. Such bacterial species are often referred
to as obligate anaerobes. A significant number of pathogenic bacteria are anaerobic. In addition to
the well-known species of Clostridium (C. tetani, C. botulinum, C. histolyticum etc.}, there are
many anaerobic and microaerophilic rods and cocci that are frequently encountered in clinical and
soil samples.
Thioglycolate Medium:
Thioglycolate medium is a nutrient medium that contains thioglycolic acid . This compound is a
::ery strong reducing agent that reacts quickly with any oxygen that may diffuse into the medium;
~owever, once the thioglycolic acid has been saturated with oxygen, the medium is no longer
Jseful for the cultivation of anaerobes.
Thioglycolate medium is typically used as a semi-solid, tubed medium and is inoculated with
a single stab into the center of the deep. It may, however, be used as a broth, or may be solidified
.vith additional agar and used in plates. The use of a small amount of agar to make it semi-solid
~as the advantage of reducing the diffusion rate.
Dehydrated Culture Media
Most essential culture media are available commercially in ready-made dehydrated form. Dehydrated
culture media are complex combinations of carefully selected components milled to produce very fine
particles of uniform size. These are carefully blended to give a balanced stable product of superbly
consistent high quality thus ensuring maximum recovery of microorganisms.
Dehydrated culture media should be stored at cool even temperatures in their closed containers in a
dry location away from direct light. Once a container is opened it should be used within 6 months .
Dehydrated culture media should be rehydrated and prepared according to the directions specified on the
package label.
Most culture media are available in a dehydrated powder form in the market. These are commercial
preparations and used after weighing and dissolving in required quantity of distilled water. Instructions for
the preparation are generally mentioned on the label of container. It is necessary to weight them accurately
and prepare them according to direction on the label.
i. Preparation of Nutrient Broth
Nutrient broth forms the basis of most media used in the study of the common pathogenic bacteria.
Nutrient broth is used to support the growth of microorganisms that have no special nutritional requirements.
Nutrient broth is most economically prepared from dehydrated media, available from different manufacturers
and is prepared and sterilized according to instruction provided by manufacturer. It is a type of liquid
media devoid of agar. The requirements of nutrients is met by supplementing beef extract and peptones:
Requirements
Conicat flask
Measuring cylinder
Test tube
Weighing machine
Autoclave
Nutrient broth powder
Cotton plug
Heater
Procedure
1. Accurately weigh the required amount of Nutrient broth powder according to instruction and transfer
it into a conical flask containing 500 ml distilled water.
2. Add more distilled water to make the volume to 1 liter.
3. Gently heat the contents with slight agitation to dissolve the ingredients .
4. Dispense 5 ml to10 ml broth to each culture tubes with the help of pipette.
5. Prepare cotton plugs and apply them into the mouth of broth tubes.
6. Transfer all the broth tubes into a test tube stand .
7. Place the stand inside the autoclave and sterilize at 121 °C, under 151bslinchpressure for 15
minutes.
8. After autoclaving, when temperature cools down , take out the broth tubes.
9. You can use the Nutrient Broth immediately or store for further use in a refrigerator.
