INTRODUCTION TO MICROBIOLOGY II

(MCB 202)

LECTURE ONE

INSTRUCTORS :
PROF. B.O. OMAFUVBE (Coordinator)
PROF. D. A. AKINPELU
PROF (MRS) A. O. OLUDURO
 LABORATORY
METHODS INVOLVED
IN THE ISOLATION,
CULTIVATION,
PRESERVATION AND
IDENTIFICATION OF
MICROORGANISMS.

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 ISOLATION
Microorganisms are ubiquitous
in nature (found everywhere)
and so they can be isolated from
diverse sources. To isolate
microorganisms from the
environment, diluents and
culture media are required.
 DILUENTS

• Physiological saline solution • Azide saline solution composing of

consisting of 0.85% NaCl in H2O. sodium azide(0.08%) in either
physiologicalsaline solution or
• Phosphate saline solution consisting phosphate saline solution.
of 0.8 g NaCl, 0.034 KHPO4 and
0.121 diKHPO4 made up to 100 mL. • Maximum Recovery Diluent (MRD)
composed of 1.0 g peptone and 8.5 g
• Ringer’s solution composing of NaCl in 1 litre of distilled water.
NaCl, KCl. CaCl, NaHCO4 and MRD is the most commonly used
distilled water. diluent.
• Peptone water composing of • The type of diluent used depends on
Bacteriological peptone and NaCl the organism because some diluent
may destroy or kill some organisms.
• Borate: Calcium saline composing of
NaCl, CaCl, borate and water • Note that diluents must not be used
direct from a refrigerator because
cold shock may prevent the
organisms from reproducing.

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 CULTURE MEDIA

Culture medium is a nutrient

material prepared for the growth
of microorganisms in the
laboratory. Some organisms can
grow well on just any culture
medium, others cannot grow on
any nonliving medium yet
developed (they are called
non-culturable organisms) while
others require special media.
 CULTURE MEDIA CONTD.
• When microorganisms are introduced into a culture medium to initiate growth,
they are called inoculum, the microbes that grow and multiply in or on a
culture medium is referred to as culture.
• A wide variety of media are available for the growth of microorganisms in the
laboratory. Most of the media available from commercial sources, have
premixed components and require only the addition of water and then
sterilization
• Media are constantly being developed or revised for use in the isolation and
identification of bacteria that are of interest to researchers in such fields as
food, water and clinical microbiology.
• When it is desirable to grow microorganisms on a solid medium, a solidifying
or gelling agent such as agar is added to the medium. Other gelling agents
available are gelatin, alginate. Agar is a complex polysaccharides derived from
a marine alga and has long been used as a thickener in food such as jellies and
ice cream.

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 CULTURE MEDIA
Important properties of agar include the following;
i. It is not an inert constituent of culture media.
ii. It can contribute metals, minerals, sulphate and pyruvate and can bind water
and inhibit growth of organisms.
iii. Few microorganisms can degrade agar, so it remains solid.
iv. Agar liquifies at about 100oC and remains liquid until the temperature drops
to about 40oC.
v. For laboratory use, agar is held in water baths at about 50oC, at this
temperature, it doesn't injure most bacteria when it is poured.
vi. Once the agar has solidified, it can be incubated at temperatures approaching
100oC before it again liquifies - this property is particularly useful for the
growth of thermophilic bacteria.

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 • Culture medium is used in the
COMPOSITION OF laboratory for the growth of
microorganisms. The medium may
CULTURE MEDIA resemble the natural substrate on
which the microorganisms usually
grow (e.g. blood serum for animal
pathogens, milk for milk organisms,
soil extract for soil microorganisms).
For a nutrient medium to fulfil its
purpose it must contain (in
appropriate proportions) all the
substances or compounds necessary
for the growth and reproduction of the
organisms.

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COMPOSITION OF CULTURE MEDIA
• Due to the diversity of microorganisms and their diverse metabolic pathways
there are numerous media. In general culture media will contain the following:
✔ Water: all life is dependent on the presence of water and all the nutrients from which
microorganisms synthesize cell material and obtain energy must be dissolved in water.
✔ Amino-nitrogen nutrients: these are protein hydrolysates (peptones) infusions or
extracts or other nitrogen-containing inorganic salts e.g K and Na nitrates. These
nutrients contain sufficient energy-rich molecules and trace elements.
✔ Energy sources: This is usually carbohydrate (glucose), but other easily used
carbohydrate may be substituted.
✔ Accessary growth factors: Some more fastidious organisms require heat-labile
supplements to stimulate growth e.g. blood, serum and vitamin complexes (Thiamine
biotin and nicotinic acid).

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 COMPOSITION OF CULTURE MEDIA
✔ Buffer salts: Soluble sodium or magnesium phosphate, acetate or citrate salts are
commonly added to culture media containing carbohydrates to maintain pH stability
when fermentative organisms are growing.
✔ Mineral salts and metals: Supplements of salts and metals are normally restricted to
synthetic (defined) culture media. Undefined media provide an adequate supply.
✔ Selective agents or components: Toxic chemicals, antibiotics and inhibitory dyes have
been used separately or in combination as culture media to select for a particular group
of organisms while suppressing the growth of the background microbiota e.g. bile salts,
selenite, tellurite dyes (eosin) etc.
✔ Indicator dyes: Dyes such as phenol red, neutral red and bromocresol purple are added
to culture media to indicate changes in pH value, during and after growth. Fermentative
carbohydrates are normally added to the formulation.

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COMPOSITION OF CULTURE MEDIA
• Solidifying agent (Agar): Concentration of 15 g/L are used to form solid
media while lower concentrations of 7.5 g/L are used to produce soft or
semisolid media. Depending upon the composition of the culture media,
they may be divided into two major groups namely:
o Defined (synthetic) media: these are chemically defined media with defined
chemical composition. They are not practical for use in most routine lab work but
valuable for nutritional studies.
o Complex media: these contain essentially not well defined components such as
peptone, beef or malt extracts or blood. Commonly used complex medium is
Nutrient agar/Nutrient broth.

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COMPOSITION OF CULTURE MEDIA
• Depending on the nutritional requirement of a particular
microorganism, culture media may be differentiated into minimal
(incomplete) medium or complete medium.
• A minimal medium contains only the components which satisfy the
minimum nutritional requirement of microorganisms. It is always a
defined medium and mostly used in the maintenance of
microorganisms in culture.
• A complete medium consists of not only all essential nutrients but
also components (or precursors) of substances which the organisms
can produce to enhance their growth

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CLASSIFICATION OF MEDIUM BASED ON THEIR
COMPOSITION OR THE GROUP OF ORGANISMS THEY CAN
SUPPORT

NON SELECTIVE
SELECTIVE MEDIUM DIFFERENTIAL MEDIUM
CULTURE MEDIUM

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CLASSIFICATION OF MEDIUM BASED ON THEIR
COMPOSITION OR THE GROUP OF ORGANISMS THEY CAN
SUPPORT

ELECTIVE MEDIUM ENRICHED MEDIUM ENRICHMENT MEDIUM

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NON SELECTIVE CULTURE MEDIUM

• This is a medium used for the

cultivation of common or most
bacteria. It is sometimes called
Routine laboratory medium. E.g.
Nutrient broth and Nutrient agar

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 SELECTIVE MEDIUM
• This is a basic medium which
contain selective components such
as bile salt, various dyes, high
concentration of NaCl that inhibit
the growth of non target
microorganisms and favour the
growth of specific organisms.
They are useful in the isolation of
specific microorganisms from
mixed populations.

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SELECTIVE MEDIUM

• Selective toxic compounds or • Antimicrobial agents used to

inhibitory compounds used in the suppress specific types of
preparation of selective medium microorganisms include
include Bile salts, selenite, ampicillin, chloramphenicol,
tetrathionate, tellurite, azide, colistin, cycloheximide etc. in
sodium lauryl sulfate(SLS) high some cases, combinations of
sodium chloride concentrations, antimicrobials are effective in
various dyes (eosin, crystal violet suppressing classes of
and methylene blue). microorganisms

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SELECTIVE MEDIUM
Selective agar or medium include Selective agar or medium include

• MacConkey agar: containing bile • Selective media for the isolation

salts and crystal violet dye which of Gram positive bacteria –
inhibit the growth of Gram Potassium tellurite, thallium
positive bacteria and thus acetate and Sodium azide added
selective for Gram negative and to media inhibit the growth of
enteric bacteria. Gram negative bacteria thus
selective for Gram positive
bacteria.

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DIFFERENTIAL MEDIUM

• Differential medium is one in

which certain species of microbes
produce characteristic colonies
which can easily be recognized.
The differentiation of many
microorganism is based upon the
production of acid from various
carbohydrates and other carbon
sources or the decarboxylation of
amino acids.

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DIFFERENTIAL MEDIUM

S/No pH indicator Acid colour Alkaline colour

• Some media include pH indicators,
that permits the visual detection of I Methyl red Yellow Red

changes in pH resulting from Ii Bromocresol purple Yellow Purple

metabolic reactions.
Iii Bromothymol blue Yellow Blue

Iv Phenol Red Yellow Red

v Cresol Red Yellow Red

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DIFFERENTIAL MEDIUM

• Examples of differential medium is Blood agar and MacConkey agar

• On blood agar, haemolytic and non-haemolytic species can be
differentiated. On MacConkey agar, lactose fermenting coliforms
produce red colonies (as a result of acid production affecting the neutral
red indicator), while non-lactose fermenting intestinal organisms
produce colourless colonies. E.g Salmonella sp.

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ELECTIVE MEDIUM

• Elective medium is designed to have minimal nutritional ingredients,

enabling only a limited group of microorganisms to grow.
• An elective media may be very useful, particularly if the required
organisms have unusual nutritional characteristics. E.g. Wild yeasts can
be isolated using lysine agar on which organisms cannot grow unless
they can use lysine as a sole source of nitrogen.
• Elective media are not used in medical microbiology because they are
not effective for resuscitation of stressed organisms.

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ENRICHED MEDIUM

• Enriched medium is one which

contain certain substances such as
blood or serum, necessary for the
growth of some fastidious
organisms.

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ENRICHMENT MEDIUM

• Enrichment medium contains substances capable of inhibiting the

growth of one kind of organism whilst allowing unrestricted growth of
the desired organism which may be present in small number, thus
enriching the population. For example, in selenite broth, Salmonella and
some strains of Shigella will grow but most of the coliform and other
bacteria which are inhibited by selenite will not. Hence it is used in the
enrichment and subsequent isolation of these organisms on solid agar
from faeces or other sources suspected of faecal contamination.
• NOTE: Enrichment takes place in liquid media (broth).

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PREPARATION OF
CULTURE MEDIA
• Media constituents and complete
culture media are sold in dehydrated
form.
• The dehydrated medium is dissolved
in distilled water to the stipulated
concentration (following the
manufacturers recommendation or
instructions) and then sterilized.

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PREPARATION OF CULTURE MEDIA

• Dehydrated medium containing agar is heated gently, usually to boiling,

to dissolve the agar.
• Many media are sterilized by exposure to elevated temperatures. The
most common method is to autoclave the medium. Different sterilization
procedures are employed when heat labile compounds are included in
the formulation of the medium.

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PREPARATION OF CULTURE MEDIA

• Autoclaving uses exposure to steam, generally under pressure to kill

microorganisms. Exposure for 15 mins to steam at 15 psi-121 oC is most
commonly used which kills both vegetative and bacterial endospores.
• Lower temperatures and different exposure times are sometimes used or
employed when components of the medium do not tolerate high
temperature. For example, media containing CHOs are often sterilized at
116oC – 118oC to prevent the decomposition of the CHOs and the
formation of toxic compounds that would inhibit microbial growth.
Other methods include filter sterilization.

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