Aula 3 Ok

Cultura de células e tecidos
47058
Cláudia Oliveira | csoliveira@ua.pt 2018_2019

Aula 4
Cultura de Células e Tecidos› 47058
2018-2019
The Microbial World
What are microorganisms?

 Microscopic forms of life (too small to see with unaided eye)
 Usually consist of single cell organisms
 Ubiquitous
 Both helpfull and problematic
http://samples.jbpub.com/9781284023473/9781284057164_CH01_Secure.pdf
2018-2019
The Microbial World
What are microorganisms?

 Microscopic forms of life (too small to see with unaided eye)
 Usually consist of single cell organisms
 Ubiquitous
 Both helpfull and problematic
In a drop (1mL) of seawater, one can find 10 million viruses, 1

million bacteria and about 1,000 small protozoans and algae
(called “protists”).
https://dornsife.usc.edu/assets/sites/378/docs/Caron_pdfs/2016_FuhrmanCaron_MEM4thEd.pdf
2018-2019
The Tree of Life
2018-2019
The Tree of Life
2018-2019
Microbial dimensions
2018-2019
Viruses
 No cellular organization
 Genetic information surrounded by a coat of protein
Viruses  Replicate actively inside living (host) cells
 Cause infectious diseases (hepatitis, influenza, AIDS…)

 Viral therapy to convert them into therapeutic agents by reprogramming to
treat diseases (anti-cancer oncolytic viruses, viral vectors for gene therapy and
viral immunotherapy).
2018-2019
Prokaryotic-Bacteria and Archaea
• Wide range of metabolic capabilities and therefore found in a variety of

(extreme) environments
• Can be photosynthetic (Cyanobacteria) or non-photosynthetic (obtain
Bacteria energy from organic or inorganic compounds, )
• Biofilm or planktonic lifestyle
• Growth in the presence of air (aerobic) or in its absence (anaerobic) or
can be facultative anaerobic
Celular morphologies
2018-2019
Prokaryotic-Bacteria and Archaea
• Lesser range of metabolic capabilities than bacteria and more than

Eukaryotes
• Morphological identical with bacteria (with exceptions, such as
Archaea Haloquadratum walsbyi)
• Genes and several metabolic pathways more closely related to those of
Eukaryotes (such as enzymes involved in transcription and translation)
• No known species forms spores
 Initially thought to be exclusive from extreme environments, but are ubiquitious

 No association with pathogenicity
2018-2019
Haloquadratum walsbyi
2018-2019
Archaea
• Methane producing archaea are used for biogas production and sewage
Biotechnology treatment
• Use of enzymes from extremophiles that can endure high temperatures
and organic solvents-green chemistry to synthesize organic compounds
2018-2019
Protists: protozoa and algae
• Do not form a natural group, or clade

• Unicellular or unicellular-colonial which form no tissues (according to whitaker)
Protists • Heterotrophic and sometimes parasitic
• Flagellata, Ciliophora, amoeba, and Sporozoa
• All types of nutrition
Nutritional types in protist metabolism

Nutritional type Source of energy Source of carbon Examples
Organic compounds or
Photoautotrophs Sunlight Most algae
carbon fixation
Chemoheterotrophs Organic compounds Organic compounds Apicomplexa, Trypanosomes or Amoebae
2018-2019
Fungi (molds, mushrooms and yeasts)
 Multicellular fungi form filaments, yeasts grow unicelularly

 Major decomposers
Fungi  Simbiontes and parasites
 High biotechnological potential (antibiotics, detergents, enzymes…)
 Over 100,000 species of fungi

 4 phyla (classified acording to their reproductive structures)
2018-2019
Fungi (molds, mushrooms and yeasts)
 Over 100,000 species of fungi

 4 phyla (classified acording to their reproductive structures)
Spores, hyphae and mycelia
2018-2019
Microorganisms must be removed from natural

environments and cultured in the laboratory. This
requires artificial media and surfaces on which they may
grow.
Knowledge of nutritional and environmental
requirements (such as temperature of incubation and/or
oxygen).
Microorganisms of interest must be separated from all

others in the environmental sample.
This requires separation techniques that allow isolation
of a pure culture of one type.
2018-2019
A pure culture is achieved, no

contaminating can be introduced from
the environment.
This requires that all media and lab
supplies be sterile.
Techniques are needed that facilitate

working with pure cultures. This
requires aseptic technique and
techniques of storage for pure cultures.
2018-2019
Culturing viruses
 Since the viruses are obligate intracellular parasites, they cannot be

grown on any inanimate culture medium
 Viruses can be cultivated within suitable hosts, such as a living cell
 There is no universal cell that will support all viruses

 Viruses tend to be host specific; therefore:
 human viruses grow best in cells of human origin,
 bovine viruses in bovine cells,
 Phages in bacterial cells
 while some viruses will not grow in vitro at all
 …
 In the laboratory the suspected virus must be grown using

a culture method known to support its growth
2018-2019
Culturing protists
 Environmental forms of some protozoan parasites, such as

spores and oocysts, are cultivable in susceptible host cells
There are some general methods of cultivation:

-Monoxenic cultures often required (interference in biochemical study…)
-Often it is required a period of accommodation for domestication of laboratory strains.
-It is wise to take note of the temperature and pH of the collecting site and, if possible,
to bring back water with which culturing can begin
http://online.sfsu.edu/antipa/biol450/handouts/appendix.pdf
2018-2019
Culturing protists
 Environmental forms of some protozoan parasites, such as

spores and oocysts, are cultivable in susceptible host cells
Animals used for in vivo cultivation of parasites
There are some general methods of cultivation:

-Monoxenic cultures often required (interference in biochemical study…)
-Often it is required a period of accommodation for domestication of laboratory strains.
-It is wise to take note of the temperature and pH of the collecting site and, if possible,
to bring back water with which culturing can begin
2018-2019
Culturing protists
 The progressive refinement of culture conditions includes

switching from the water of collection to defined
synthetic salt solutions and, in the case of phagotrophic
organisms, from an undefined diet to a specific one.
Media for cultivation of luminal parasitic protists
2018-2019
Culturing protists
 For other waterborne parasites, such as Giardia lamblia and Cyclospora cayatenensis, culture
from the environmental stage (the cyst or oocyst) is still a challenge
Giardia lamblia
Axenic culture of unicellular protists in marine broth Axenic culture of Euglena spirogyra
2018-2019
Culturing fungi
A variety of media types:
 Solid or liquid
 Most grow in medium containing a high carbohydrate source, nitrogen source, a pH of 5-6, and a
temperature range from 15-37 C.
 Some cannot be cultured in vitro, and survive only on their plant host (example: Plasmopara
viticola, the causal agent of grapevine downy mildew)
 Natural and synthetic.
Herbaceous or woody stems,

Contain defined amounts of carbohydrates,
seeds, leaves, corn meal, wheat
nitrogen, and vitamin sources (Czapek-Dox
germ, and oat meal:
medium, glucose-asparagine and Neurospora
(meal agar, potato dextrose
crassa minimal medium)
agar…)
2018-2019
Culturing bacteria (the example of)
The requirements for growth

2018-2019
Culturing bacteria: culture media pH
 Optimum pH: the pH at which the microorganism grows best

 Most grow between pH6.5 and 7.5
 They can be classified according to their pH tolerance.
2018-2019
Culturing bacteria: temperature

2018-2019
Culturing bacteria: temperature

2018-2019
Culturing bacteria: oxygen use

2018-2019
Culturing bacteria: culture media
Classification according :
- Physical state
- Chemical composition
- Function types
CULTURE MEDIA• Used to grow bacteria• Can be used to: • Enrich the numbers of
bacteria • Select for certain bacteria and suppress others • Differentiate among
different kinds of bacteria
2018-2019
Culturing bacteria: culture media
Physical state
• Liquid media: e.g. Nutrient broth

• Semi-solid media
• Solid media: e.g Nutrient agar
Solid media contain a high percebt (1-5%) of agar,

which enables the formation of discrete colonies
2018-2019
Culture media: agar

2018-2019
Culturing bacteria
2018-2019
Culture media: chemical composition
• Natural (empirical) media. E .g. juices, milk, blood
• Semi-Synthetic (complex): the medium has natural

components (unknown chemical composition) and certain
specific nutrients. E.g Tryptic Soy
• Synthetic Media (chemical defined media). All chemical

of known composition are mixed in define proportions.
(E.g Chromocult)
2018-2019
Culture media: chemical composition

2018-2019
Culturing media: functional types
Selective media
Differential media
Enriched media
2018-2019
Culturing media: functional types

2018-2019
Culturing bacteria
2018-2019
Culturing bacteria
To better characterize the culture/to confirm:

-Biochemical markers
-Genetic and molecular markers

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Cultura de células e tecidos

Cláudia Oliveira | csoliveira@ua.pt 2018_2019

The Microbial World

What are microorganisms?

The Microbial World

What are microorganisms?

In a drop (1mL) of seawater, one can find 10 million viruses, 1

The Tree of Life

The Tree of Life

 Cause infectious diseases (hepatitis, influenza, AIDS…)

Prokaryotic-Bacteria and Archaea

• Wide range of metabolic capabilities and therefore found in a variety of

Prokaryotic-Bacteria and Archaea

• Lesser range of metabolic capabilities than bacteria and more than

 Initially thought to be exclusive from extreme environments, but are ubiquitious

Protists: protozoa and algae

• Do not form a natural group, or clade

Nutritional types in protist metabolism

Chemoheterotrophs Organic compounds Organic compounds Apicomplexa, Trypanosomes or Amoebae

Fungi (molds, mushrooms and yeasts)

 Multicellular fungi form filaments, yeasts grow unicelularly

 Over 100,000 species of fungi

Fungi (molds, mushrooms and yeasts)

 Over 100,000 species of fungi

Spores, hyphae and mycelia

Microorganisms must be removed from natural

Microorganisms of interest must be separated from all

A pure culture is achieved, no

Techniques are needed that facilitate

 Since the viruses are obligate intracellular parasites, they cannot be

 There is no universal cell that will support all viruses

 In the laboratory the suspected virus must be grown using

 Environmental forms of some protozoan parasites, such as

There are some general methods of cultivation:

 Environmental forms of some protozoan parasites, such as

Animals used for in vivo cultivation of parasites

There are some general methods of cultivation:

 The progressive refinement of culture conditions includes

Media for cultivation of luminal parasitic protists

A variety of media types:

Herbaceous or woody stems,

Culturing bacteria (the example of)

The requirements for growth

Culturing bacteria: culture media pH

 Optimum pH: the pH at which the microorganism grows best

Culturing bacteria: temperature

Culturing bacteria: temperature

Culturing bacteria: oxygen use

Culturing bacteria: culture media

Culturing bacteria: culture media

• Liquid media: e.g. Nutrient broth

Solid media contain a high percebt (1-5%) of agar,

Culture media: agar

Culture media: chemical composition

• Natural (empirical) media. E .g. juices, milk, blood

• Semi-Synthetic (complex): the medium has natural

• Synthetic Media (chemical defined media). All chemical

Culture media: chemical composition

Culturing media: functional types

Culturing media: functional types

To better characterize the culture/to confirm:

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