Transcript Culture medium

NAS:
Cultivation and Isolation of Bacteria
You’re
never too
old to
streak!
Lecturer: Mr M. Zivuku
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Definitions
• Culture A culture is the microorganisms that grow in a
culture medium.
• To culture means to grow microorganisms in a culture
medium
• Culture medium [pl. media] Culture media are
solutions containing all of the nutrients and necessary
physical growth parameters necessary for microbial
growth.
• Note that not all microorganisms can grow in any given
culture medium and, in fact, many can't grow in any
known culture medium.
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Definitions
• In addition to chemical and physical characteristics,
media can be distinguished qualitatively as:
• Solid vs broth
• Non-synthetic vs chemically defined
• Reducing
• Selective
• Differential
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Definitions
• Solid medium [agar] Solid medium is media containing
agar or some other, mostly inert solidifying agent.
• Solid medium has physical structure (broth lacks
structure) and this allows bacteria to grow in physically
informative or useful ways (e.g., as colonies or in
streaks).
• Solid medium is usually used as:
-Slants
-Stabs
-Petri dishes
• See illustration below.
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Various examples solid media
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Definitions
• Colony A colony is a pile or mass of a sufficiently large
number of cells, growing on or in solid medium, that
they are visible to the naked eye.
• Broth medium Broth medium is media lacking a
solidifying matrix (see solid medium).
• Non-synthetic [chemically undefined] medium
Chemically undefined ingredient:
– Non-synthetic medium contains at least one
component that is neither purified nor completely
characterized nor even completely consistent from
batch to batch.
– Often these are partially digested proteins from
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various organismal sources.
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-Nutrient broth, for example, is derived from cultures of
yeasts.
-May be broth or solid.
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Pure culture technique Clonal populations:
– Pure culture technique is a method of culturing
microorganisms in which all of the individuals in a
culture have descended from a single individual.
– This is done so as to:
• inhibit evolutionary change within cultures
• allow the characterization of types microorganisms
without the confounding presence of other,
different types of microorganisms
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Pure culture technique
• Colony isolation: The basis of pure culture technique is
the isolation, in colonies, of individual cells, and their
descendants, from other colonies of individuals.
• This is usually done by culturing methods employing
Petri dishes such as:
– Streaking
– Pouring
– spreading
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Pure culture techniques
• Isolation from the wild:
• When isolating microorganisms from complex mixtures it
is always a good idea to repeat the isolation procedure
at least once (e.g., restreak an isolated colony) to make
sure that an isolated colony is truly derived from only a
single cell (i.e., closely overlapping colonies can be
indistinguishable from colonies founded from single
cells)
• Following their isolation from the wild, microorganisms
may be characterized by inoculation into differential
medium to determine what type of nutrients they require
or can use, and what types of by-products they produce.
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This aids in identification
Colony morphology
• Differentiating colonies:
– Colony morphology gives important clues as
to the identity of their constituent
microorganisms.
– Important classes of characteristics include:
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size
type of margin
colony elevation
colony texture
light transmission
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colony pigmentation
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Illustration, variation in colony margins
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Streaking
• Petri dish Petri dishes are circular, vertical sided plates
used to contain agar and with tops for aseptic purposes.
• Loop A platinum wire formed into a loop is heated to an
orange glow to sterilize it then is used to transfer a
culture from one physical location to another.
• Streaking is a method of applying cultures to solid
medium: a sterile loop is cooled and brought into contact
with a culture
• the loop is then brought into contact with the surface of
solid medium whereupon it is streaked (i.e., dragged)
along the surface of the solid medium
• colonies grow along the points of the streak
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Illustration streaking a plate
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Purpose of streaking: To obtain pure, isolated colonies.
Principle: By spreading a large amount of bacteria over the
large surface area of a plate, the amount of bacteria is diluted
until individual cells are spread on the surface of the
plate. Each individual cell grows into a single colony. All of the
cells in this colony are genetically identical.
Each species of
bacteria produces a
distinctive colony
appearance.
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Nice isolation!
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Streak pattern
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Bacterial growth pattern
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Starting with a mixed
culture, isolation of
different species may be
obtained by streaking
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Example of a poorly streaked plate: Bacterial
growth is too heavy and isolation is poor.
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Contamination of a streak plate results from leaving the plate
open too long or not shielding properly with the lid.
Correct procedure
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Which streak plate culture started as a pure culture. How can you tell?
Answer: the one on the right, because all colonies look alike.
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Pour plate Method
• A pour plate is a method of melted agar inoculation
followed by petri dish incubation.
• Steps include:
– Cultures are inoculated into melted agar that has
been cooled to 45�C
– the liquid medium is well mixed then poured into a
petri dish (or vice versa)
– colonies form within the agar matrix rather than on top
as they do when streaking plate
• Pour plates are useful for quantifying microorganisms
that grow in solid medium.
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Pour plate method
• Because the "pour plate" embeds colonies in agar it can
supply a sufficiently oxygen deficient environment that it
can allow the growth and quantification of
microaerophiles
• Spreading a plate is an additional method of quantifying
microorganisms on solid medium.
– Instead of embedding microorganisms into agar, as is
done with the pour plate method, liquid cultures are
spread on the agar surface using a devise that looks
more or less like a hockey stick.
• An advantage of spreading a plate over the pour plate
method is that cultures are never exposed to 45�C+
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melted agar temperatures.
Preservation of microbes
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Methods of preserving cultures include:
Refrigeration
Stabs
Slants
Lyopholisation
freezing
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Aseptically pouring
agar plates
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Agar plates are stored upside down to
prevent condensation.
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All labeling is done on the bottom of the agar plate
1. Initials
2. Date (mm/dd/yy)
3. Code # or letter
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Environmental sampling
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Typical environmental sampling results
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Microbes in the Environment. This plate was inoculated with 500 L
of indoor air. Approximately how many colonies are on the plate?
How many different bacterial species are here?
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Microbes in the Environment. This plate was inoculated with a cotton
swab that was wiped over a shower drain.
Fungi usually produce large, "fuzzy" colonies (marked F).
Approximately how many colonies are on the plate? How many are
bacterial colonies? How many different bacterial species are here?
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