Cultivation, isolation.

Purification and
characterization of microorganisms
• A nutrient material prepared for the growth of
microorganisms in a laboratory is called a culture
medium.
• Some bacteria can grow well on just about any culture
medium, others require special media.
• When microbes are introduced into any culture
medium to initiate growth ,they are called an
inoculum.
• The microbes that grow and multiply in or on a culture
medium are referred to as a culture.
• For growing a culture of certain microorganism, perhaps the
microbes from a particular clinical specimen.
• it must contain the right nutrients for the specific
microorganisms we want to grow.
• it should also contain sufficient moisture, a properly adjusted
PH.
• The medium must initially be sterile____ that is it must
initially contain no living microorganisms__ so that the
Culture will contain only the microbes we add to the medium.
• Finally the growing culture should be incubated at the proper
temperature.
• A wide variety of media are available for the growth of
microorganisms in the laboratory.
• Most of these media which are available from
commercial sources, have premixed components and
require only the addition of water and then sterilization.
• When it is desirable to grow bacteria on a solid medium,
a solidifying agent such as agar is added to the medium.
• A complex polysaccharide derived from a marine alga,
agar has long been used as a thickener in foods such as
jellies and icecream.
• Agar media are usually contained in test tubes or
petri dishes.
• The test tubes are called slants when they are
allowed to solidify with the tube held at an angle so
that a large surface area for growth is available.
• When the agar solidifies in a vertical tube, it is called
a deep.
• Petri dishes named for their inventor are shallow
dishes with a lid that nests over the bottom to
prevent contamination, when Filled they are called
petri(or culture) plates.
 Chemically defined media
• To support microbial growth, a medium must
provide an energy source ,as well as sources of
carbon,nitrogen,sulphur,phosphorus and any
organic growth factors the organism is unable
to synthesize.
• A chemically defined medium is one whose
exact chemical composition is known.
• For a chemoheterotroph a chemically defined
medium must contain organic growth factors
that serve as a source of carbon and energy.
• For example glucose is included in the medium
for growing the chemoheterotroph E.coli.
• Many organic growth factors must be provided
in the chemically defined medium used to
cultivate a species of Neisseria.
• Organisms that require many growth factors are
called fastidious.
 Selective and differential media
• In clinical and public health microbiology, it is
frequently necessary to detect the presence of
specific microorganisms associated with disease
or poor sanitation.
• For this task selective and differential media are
used.
• Selective media are designed to suppress the growth of
unwanted bacteria and encourage the growth of desired
microbes.
• For example,
• Bismuth sulfite agar is one medium used isolate the
typhoid bacterium, gram negative salmonella typhi from
feces.
• Bismuth sulfite inhibits gram positive bacteria and most
gram negative intestinal bacteria(other than s. typhi)as
well.
• Sabouraud’s dextrose agar, which has a PH of 5.6 is used
to isolate fungi that 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.
• Streptococcus pyogenes the bacterium that causes strep
throat ,show a clear ring around their colonies.
• They are designed to differentiate among microbes.
• Different bacterial species may produce dissimilar colony
colors when grown on differential agar.
• While in differential broth cultures, the media change
color.
• Differential media are used to confirm the identity of a
microbe that has already been isolated by culturing in
enrichment and selective media.
 Complex media
• Chemically defined media are usually reserved for
laboratory experimental work or for the growth of
autotrophic bacteria.
• Most heterotrophic bacteria and fungi are routinely
grown on complex media, made up of nutrients
including extracts from yeasts, meat or plants or
digests of proteins from these or other sources.
• In complex media, the energy, carbon, nitrogen,
and Sulphur requirements of the growing
microorganisms are peptones.
• These small soluble fragments can be digested by
most bacteria.
• Vitamins or other growth factors are provided by
yeasts extracts or meat extracts.
• Yeast extracts are particularly rich in b vitamins.
 Enrichment culture
• Because bacteria present in small numbers can be
missed ,especially if other bacteria are present in much
larger numbers, it is sometimes necessary to use an
enrichment culture.
• Enrichment medium usually provides nutrients that
favor the growth of a particular microbe but not
others.
• In this sense it is also a selective medium, but it is
designed to increase very small numbers of the desired
type of organism to detectable levels.
• Suppose we want to isolate from a soil sample a
microbe that can grow on phenol and is present
in much smaller numbers than other species.
• If the soil sample is placed in an enrichment
medium in which phenol is the only source of
carbon and energy, microbes unable to
metabolize phenol will not grow.
 Obtaining pure cultures
• Most infectious materials.
• Such as pus, sputum , and urine contain several
different kinds of bacteria so do samples of
soil,water,or food.
• If these materials are plated out onto the surface of
a solid medium, colonies will form that are exact
copies of the original organism.
• A visible colony theoretically arises from a single
spore or vegetative cell or from a group of the same
microorganisms attached to one another in clumps
or chains.
• Microbial colonies often have a distinctive
appearance that distinguishes one microbe from
another.
 streaking
• The isolation method most commonly used to
get pure cultures is the streak plate method.
• A sterile inoculating loop is dipped into the mixed
culture that contains more than one type of
microbe and is streaked in a pattern over the
surface of the nutrient medium.
• As the pattern is traced, bacteria are rubbed off
the loop onto the medium.
• The last cells to be rubbed off the loop are far
enough apart to grow into isolated colonies.
• These colonies can picked up with an inoculating
loop and transferred to a test tube of nutrient
medium to form a pure culture containing only one
type of bacterium.
• The streak plate method works well when the
organism to be isolated is present in large numbers
relative to the total population.
• However when the microbe to be isolated is
present only in very small numbers, its
numbers must be greatly increased by
selective enrichment before it can be isolated
with the streak plate method.
 Streaking
• This is most widely used method of isolation.
• The technique consists of pouring a suitable sterile
medium into sterile petri plate and allowing the
medium to solidify.
• By means of a sterile loop or straight needle or a
sterile bent glass-rod a small amount of growth
preferably from a broth culture or bacterial
suspension is streaked back and forth across the
surface of agar until about one third of the diameter
of the plate has been covered.
• The needle is then flamed and streaking in done
at right angles to and across the first streak.
• This serves to drag bacteria out in a long line
from the initial streak.
• When this streaking is completed the needle is
again flamed and streaking is done at right
angles to the second streak and parallel to the
first.
 Dilution
• This method is used for the microorganisms which
cannot be easily isolated by streaking or plating
method.
• Sometimes when several organisms are present in a
mixture, with one organism predominating, the
predominating form may be isolated by this method.
• For example, when raw milk is allowed to sour at
room temperature it will, at the time of curding,
have a mixture of microorganisms with high
percentage of Streptococcus lactis .
• If 1 ml of the sour milk is taken into a tube
containing 9 ml. of sterile milk (in which no
organisms are present) then 1 ml. of this mixture is
transferred with a sterile pipette into a second
tube of sterile milk and the procedure is repeated
i.e. from second to third tube, third to fourth tube
until a series of about 10 tubes are inoculated.
• By this serial dilution, the chances are that a pure
culture of S. lactis will be obtained.
 4. Enrichment Procedure:
• This procedure involves the use of media and
conditions of cultivation which favors the
growth of the desired species.
• For example, when a man suffers with typhoid,
the intestinal discharge posses small number of
Salmonella Tryphosa when compared with E.
coli and other forms.
• A quantity of microbes can be measured with a pour plate,
spread plate.
• Measuring microbial population numbers can be achieved with
methods .
• There are two methods to prepare plates that successively
separate out individual microbes for future isolation: the pour
plate and the spread plate.
• In a pour plate the microbe sample is mixed with the liquid agar
before pouring the plate. A pour plate results in microbes
growing within and on top of the media in the plate.
• In a spread plate the microbe sample is placed in contact with the
solid agar surface. Spreading the culture over the plate results in
an even distribution of microbes on the surface of the agar.