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Artificial cultivation of bacteria,
culture media
Patrick
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Cultivation
The process of growing microorganisms inculture
involves taking bacteria from the infection site
growing them in artificial environment in thelaboratory
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Introduction
In nature, bacteria exist as mixed populations.
In the laboratory these populations must be
separated so that characteristics of individualspecies may be observed.
A number of basic techniques are used in
microbiology with this end in mind.
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Introduction
First, microorganisms must be removed fromnatural environments and cultured
this requires artificial media and surfaces on
which bacteria may grow this also requires knowledge of nutritional
requirements and environmental requirements
bacteria of interest must be separated from allother bacteria
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Introduction
This requires separation techniques that allowisolation of a pure culture
once a pure culture is achieved, it should bemaintained
This requires that all media and lab supplies besterile
techniques are needed that facilitate working
with pure cultures This requires aseptic technique and techniques
of storage for pure cultures
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Uses of media & culture media provide nutrients for the growth of
microbesCulture;
gives in vitro diagnosis of infectious diseases.
Isolating bacteria from sterile sites of the body
provides an initial step in studying morphology
and its identification.
provides antigens for developing serological
assays or vaccines.
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Uses of media & culture
For genetic studies and manipulations in vitro.
provides a reliable way estimating their
numbers (viable count).
solid media is a convenient way of separating
bacteria in mixtures.
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Classification of media
Based on three factors
consistency,
nutritional component, functional use,
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Based on Consistency
Culture media are
liquid,
semi-solid
or solid and biphasic.
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Liquid media
These are available for use in test-tubes, bottles. They also called broths (e.g nutrient broth).
Here, bacteria grow uniformly producing general
turbidity. Certain aerobic bacteria and those containing fimbriae
(Vibrio & Bacillus) are known to grow as a thin film
called surface pellicle on the surface of undisturbed
broth.
Sometimes the initial turbidity may be followed by
clearing due to autolysis (pneumococci)
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Liquid media
Mostly used when a large number of bacteria areneeded
When the number in sample is suspected to be low.
This is the practical approach in blood cultures.
It can be used to obtain viable count (dilution
methods).
However, properties of bacteria are not visible
presence of more than one type of bacteria can notbe detected.
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Solid media Any liquid medium can be converted to solid media by
adding a solidifying agents.
Agar agar;
is the most commonly used solidifying agent.
Agar, is a galactan obtained from marine algae It melts at 95oC and solidifies at 42oC,
It doesnt contribute any nutrients
it is not hydrolyzed by most bacteria It is free from growth promoting or
growth retarding substances.
it is used at 1-3% to make a solid agar medium.
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Semi-solid agar
Made by reducing the amount of agar to 0.2-0.5% useful in demonstrating bacterial motility
Also for transporting some bacteria
e.g. Stuarts and Amies media
mannitol motility medium.
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Biphasic media
Sometimes, a culture system comprises of both
liquid and solid medium in the same bottle.
This is known as biphasic medium.
The inoculum is added to the liquid medium
subcultures are made, by tilting the bottle
This allows the liquid to flow over the solid
medium This eliminates frequent opening of the culture
bottle to subculture
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Other solidifying agents Besides agar, egg yolk and serum too can be used
to solidify culture media.
While serum and egg yolk are normally liquid,
they can be rendered solid by coagulation using
heat.
Serum containing medium; Loefflers serum slope
egg containing media;
Lowenstein Jensen medium
Dorset egg medium are solidified
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Based on nutritional component
Media can be classified as simple, complex andsynthetic (or defined).
non-fastidious bacteria grow with minimal
requirements
Fastidious bacteria require extra nutrients.
Simple media; peptone water, nutrient agar
Complex media; blood agar, chocolate agar. Synthetic or defined media; Davis & Mingioli
medium are specially prepared media for
research purposes
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Based on functional use
These include
basal media,
enriched media,
selective/enrichment media,
indicator/differential media,
transport media and holding media.
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Basal media
These are basically simple media
they support growth of a broad range of
organisms
They usually have complex constituents Constituents of such media are generally defined
e.g. peptone water, nutrient broth and nutrient
agar are considered basal medium
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Enriched media
Addition of extra nutrients in the form of blood,serum, egg yolk etc, to basal medium
Enriched media are used to grow fastidious
bacteriaExamples;
Blood agar,
chocolate agar, Loefflers serum slope etc
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Selective media
These are designed to inhibit unwanted
organisms or contaminating bacteria
this helps to recover pathogen from a mixture of
bacteria.
selective media are agar based
addition of certain inhibitory agents to agar
media makes it selective
These should not affect the pathogen in choice
Examples; antibiotics, dyes, chemicals, alteration
of pH or a combination of these.
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Enrichment media
These are liquid media that also serves to
inhibit commensals in the clinical specimen.
Examples;
Selenite F broth, tetrathionate broth
alkaline peptone water
These are used to recover pathogens fromfecal specimens.
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Differential or indicator media Certain media are designed to different bacteria
on the basis of their colony colour.
by incorporating dyes, metabolic substrates etc,
bacteria that utilize them appear as differently
coloured colonies
Such media are called differential media or
indicator media
Examples: MacConkeys agar, CLED agar, TCBS
agar, XLD agar etc
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Transport media Clinical specimens must be transported to the
laboratory immediately after collection
to prevent overgrowth of contaminating
organisms or commensals.
This can be achieved by using transport media.
Such media prevent drying of specimen,
They also maintain the pathogens they inhibit overgrowth of unwanted bacteria.
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Transport media
Some of these media (Stuarts & Amies) are
semi-solid in consistency
Addition of charcoal serves to neutralize
inhibitory factors
Cary Blair medium is used to transport samples
from cholera suspects
Sachs buffered glycerol saline is used to
transport samples of bacillary dysentery suspects
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Anaerobic media
Anaerobic bacteria need special media ( no oxygen) and
extra nutrients
They need to be supplemented with nutrients like
hemin and vitamin K
Boiling the medium expels any dissolved oxygen
Addition of 1% glucose, 0.1% thioglycollate, 0.1%
ascorbic acid, 0.05% cysteine or red hot iron filings can
render a medium reduced
Robertson cooked meat that is commonly used to grow
Clostridium spps
It contains heart meat and nutrient broth.
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Anaerobic media
Before use the medium must be boiled in water
bath to expel any dissolved oxygen and then
sealed
Methylene blue or resazurin is an oxidation-
reduction potential indicator
it is incorporated in the thioglycollate medium
Under reduced condition, methylene blue is
colourless
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Use of physical separation procedures
Streak plate tech
For achieving single colony characteristics
streak plate technique
pour plate;
spread plate;
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Growth conditions
Bacteria may be isolated from a variety ofenvironments
For cultivation of bacteria in the lab, the
conditions of the environments must bemimicked
Refer to previous lecture on growth
requirements
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Bacterial requirements Water
Oxygen
Carbon dioxide
Temperature
PH Light
Nutrients
Growth factors Inorganic salts
Osmotic effect
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Salt conditions Halophiles; these bacteria require relatively high
concentrations of salt for growth (10-20%);
Halophiles must possess special membranes and
enzyme
Some organisms are salt tolerant. These do notrequire salt for growth but can grow in its presence
An example is Staphylococcus aureus. which is found
on skin, which often has a high salt concentration(10% NaCl).
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Preparation and preservation
Heat stable ingredients Care must be taken to adjust the pH of the
medium before autoclaving.
Dehydrated media are commercially available These must be reconstituted as per
manufacturers recommendation.
Most culture media are sterilized by autoclaving.
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Heat labile ingredients
like glucose, antibiotics, urea, serum, blood are not
autoclaved.
These are filtered and added separately after the
medium is autoclaved.
a representation from each lot is tested forperformance and contamination before use.
Once prepared, media may be held at 4-5oC in the
refrigerator for 1-2 weeks.
Certain liquid media in screw capped bottles or tubes
or cotton plugged can be held at room temperature for
weeks.
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Thank you