THE ADDITIONAL FEATURES OF SIX “I’s” AND MEDIA THE

FOUNDATION OF CULTURING
Lesson Proper
1. The uncultured
2. Inoculation, Growth, and Identification of
cultures
3. Isolation Techniques
4. Identification techniques
5. Methods of isolating bacteria
6. Types of media
7. Physical state of media, and chemical
content of media
8. Functional types of media
OVERVIEW
Millions of medical tests take place every day
across the globe. Many tests require a sample of
blood, urine, or other bodily fluid or tissue. This
required to grow the bacteria that a doctor is
looking for within a patient’s body. Interestingly,
growing bacteria is not the same as growing, for
example, a plant. However, there is one important
similarity. This similarity is known as food, or
microbiological food.
Learning outcomes
• Define inoculation, media, and culture and
describe sampling methods and instruments
and what events must be controlled.
• Describe three basic techniques for isolation
including tools media, incubation, and outcome.
• Explain what an isolated colony is and indicate
how it forms.
• Differentiate between a pure culture, sub
culture mixed culture, and contaminated
culture. Define contaminant.
• What kind of data are collected during
information gathering.
• Describe some of the process involved in
identifying microbes from samples.
• Explain the importance of media for culturing
microbes in the laboratory.
• Name the three general categories of media,
based on their inherent properties and uses.
• Compare and contrast liquid, solid, and
semisolid media, giving examples.
• Analyze chemically defined and complex
media, describing their basic differences and
content.
• Describe functional media; list several
different categories, and explain what
characterizes each type of functional media.
• Identify the qualities of enriched, selective, and
differential media; use examples to explain
their content and purposes.
• Explain what it means when microorganisms
are not culturable.
• Describe live media and the circumstances that
require it.
ADDITIONAL FEATURES
OF THE SIX “I’S”
THE UNCULTURED
• For some time, microbiologists have suspected
that culture-based methods are unable to
identify many kinds of bacteria. This was first
confirmed by environmental researchers, who
came to believe that only about 1%(and in some
environments could be grown in laboratories by
the usual methods and therefore, were unknown
and unstudied. These microbes are termed
viable but nonculturable, or VBNC.
• In 1990s, a number of specific, nonculturing
tools based on genetic testing had become
widely available. When these methods were
used to sample various environment, they
revealed a vast “jungle” of new species that
had never before been culture. These were
the techniques that Venter's team applied to
ocean sample.
INOCULATION, GROWTH, AND IDENTIFICATION
OF CULTURES
• To cultivate, or culture, microorganisms, one
introduces a tiny sample (the inoculum) into a
container of nutrient of medium, which
provides an environment in which they multiply.
This process is called Inoculation.
• The observable growth that later appear in
medium in or on the medium is known as
culture.
• The nature of the sample depends on the
objectives of the analysis.
• Clinical specimens for determining the cause of
an infectious disease are obtained from the
body fluids (blood, cerebrospinal fluids),
discharges (sputum, urine,feces)
Isolation Techniques
 Certain isolation techniques are based on the
concept that if an individual bacterial cell is
separated from the other cells and provided
adequate space on a nutrient surface, it will grow
into a discrete.
 Stage in the formation of an isolated colony,

showing the microscopic events and the

macroscopic results.
 Separation techniques such as streaking can be

used to isolated single cells. After a numerous cell

divisions, a macroscopic mound of cell, or a
colony will be formed.
 Mound of cell is called Colony(figure 3.17).
Because it arises from a single cell or clusters of
cells, an isolated colony consist of just one
species.
 Proper isolation requires that a small number of

cells be inoculated into a relatively large volume or

over an expensive area of medium. It generally
requires the following materials: a medium that has
a relatively firm surface contained in a clear, flat
covered plate called a Petri dish and inoculating
tools.
ISOLATION TECHNIQUES

Figure 3.17
 Methods For Isolating Bacteria

 Streak plate method- a small droplet of sample

is spread with a tool called inoculating loop over
the surface of the medium according to a pattern
that gradually thin out the sample and separates
the cells spatially over several section of the
plate(figure 3.18a, b)
 Loop dilution, or pour plate technique- the

sample is inoculated , also with a loop, into a

series of cooled but still liquid agar tubes so as to
dilute the number of cells in each successive tube
in the series(figure 3.18c, d). Inoculated tubes
are then plate out (pour) into sterile petri dishes
and are allowed to solidify (harden).
 Spread plate method – a small volume of liquid, a
diluted sample pipetted onto the surface of the
medium and spread around evenly by sterile
spreading tool (sometimes called “hockey stick”). As
with the streak plate, cells are spread over separate
areas on the surface so that they can form individual
colonies(figure 3.18e,f).
Methods For Isolating Bacteria
 Once a container of medium has been inoculated, it
is incubated in a temperature control
chamber(incubator) to encourage microbial growth.
Although microbes have adopted to growth
temperature ranging from freezing to boiling, the
usual temperature used in the laboratory propagation
fall between 20 ͦC and 40 ͦC. incubators can also
control the content of the atmospheric gases such as
oxygen and carbon dioxide that may be required for
the growth of a certain microbes.
 During the incubator period(ranging from a few hours

to several weeks) the microbes multiple and

produces a culture a culture with macroscopically
observable growth. Microbial growth in a liquid
medium materialize as cloudiness, sediments a
Surface mat or colored pigment.

In some ways, culturing microbes is analogous to

gardening.
1. A pure culture (axenic) – a container of medium
that grows only a single known species or a type of
microorganism. A standard method fro preparing a
pure culture is to use a subculture technique to
make a second-level culture.
2. Mixed culture – different species of organism.
3. Contaminated culture – (unwanted microbes of
uncertain identity).
 Identification Techniques
 How does one determine what sort of microorganism

have been isolated in cultures? Certainly microscopic

appearance can be valuable in differentiating the
smaller, simpler prokaryotic cells from the larger, more
complex eukaryotic cells. Appearance can often be
used to identify eukaryotic micro organisms to the
level of genus or species because of their more
distinctive appearance.
 Bacteria are generally not as readily identifiable by

these methods because of very different species may

appear quite similar. For them we must include other
techniques, some of which characterize their cellular
metabolism. These called biochemical test.
(fundamental chemicals like nutrients).
MEDIA THE FOUNDATIONS
OF CULTURING
 A major stimulus to the rise of
microbiology in the late 1800s was the
development of techniques for growing microbes
out of their natural habitats and in pure form in
laboratory.
The milestone enabled the close examination of
microbe and its morphology, physiology, and
genetics.
It was the evident from the very first that
for successful cultivation, the microorganisms
being cultured had to be provided with all of their
required nutrients in an artificial medium .
 Some microbes require only a very few simple
inorganic compounds for growth; others need a
complex list of specific inorganic and organic
compounds. This tremendous diversity is evident in
the types of media that can be prepared. At least
500 different types of media are used in culturing
and identifying microorganisms. Culture media are
contained in the test tubes, flasks, or Petri dishes,
and they are inoculated by such tools as loops,
needles, pipettes, and swabs.
 Types of Media
Media -
Three general categories based on their
properties:
Physical State Chemical Functional type (Purpose of
(Medium’s normal composition (Type Medium)
consistency) of chemicals
mediums contain
1. Liquid 1. Synthetic 1. General purpose
(chemically 2. Enriched
defined) 3. Selective
2. Semisolid 2. Nonsynthetic 4. Differential
(complex; not 5. Anaerobic growth
chemically defined 6. Specimen transport
3. Solid (can be 7. Assay
converted to liquid) 8. Enumeration
Physical States of Media
Liquid media – no agar.
 For inoculum preparation, blood culture, for the

isolation of pathogens from a mixture.

 Eg: Nutrient broth

Semi-solid media contain a low percentage (1%) of

agar, which can be
use for motility testing
Solid media – 1 to 5% agar
 provide firm surface on which cells can form

discrete colonies and are advantageous for

isolating and culturing bacteria and fungi.
 Two forms; liquefiable and nonliquefiable
 Liquefiable solid media; gelatin
 Nonliquefiable solid media - do not melt they

include material such as rice grain (used to grow

fungi) cooked meat media (good for anaerobes),
and egg or serum media that are permanently
coagulated or hardened by moist heat.
Chemical content of media
Synthetic – a Media with a chemically defined
composition, such media contain pure chemical
nutrients that vary little from one source to another
and have a molecular content specified by a means
of exac formula(protozoan Leishmania 75different
chemicals).
Nonsynthetic, or complex, medium. The
composition of this type of medium is not defiable by
an exact chemical formula. Substance that can make
it nonsynthetic are from animal or plant tissue.(blood,
serum, meat extracts, infusions, milk, soybean
digests, and peptone
Media to Suit Every Function
Comparison of selective and
differential media with
general-purpose