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cultivation
Culture media
Identification and;
susceptibility testing to antimicrobial drugs;
Conditions
In order to grow bacteria under
laboratory conditions it is essential to fulfill
two requirements:
suitable nutrients must be supplied;
the physical conditions must be as near
optimum as possible for the organism
under consideration.
BACTERIAL NUTRITION
Nutrition = the ways by which an organism
assimilates from the environment the
substances necessary for its metabolism.
Nutrients = substances for which their solutions
can cross the cytoplasmic membrane to be used
in the cells metabolism.
Bacteria feed by absorption.
In bacteria, the digestion is extracellular.
BACTERIAL NUTRITION
The main elements required for growth are
carbon, hydrogen, oxygen and nitrogen, with
sulfur and phosphorus required in somewhat
smaller amounts, and other elements such as
sodium, potassium, magnesium, iron and
manganese in considerably smaller amounts.
Hydrogen and oxygen can be supplied in the
water that is essential for any growth.
1. Energy source
Fototrophic bacteria use the energy of light.
Chemotrophic bacteria use the energy produced
by chemical oxidation reactions. These reactions
use an electron donor (the energy source) and a
electron acceptor.
* The electron donor.
The chemotrophic bacteria are litotrophs (the
electron donor is an inorganic compound) or
organotrophs (the electron donor is an organic
compound).
** The electron acceptor.
Classification of bacteria
Bacteria that use fermentation are called
anaerobes;
those which grow both in the presence or
in the absence of the oxygen are called
facultative anaerobes.
Microaerophilic bacteria grow only at low
concentrations of oxygen (5%).
2. Carbon source
Autotrophic bacteria use CO2 as a carbon
source and heterotrophic bacteria,use an
organic compound (carbohydrate or aminoacid),
also used as energy source.
Heterotrophic bacteria which need for growth
CO2 concentrations bigger than those in
atmosphere (ex. 3-5%) are called carboxyphilic
bacteria or capnophilic.
3. Nitrogen source
The main inorganic form of nitrogen used
in biosynthesis is ammonia, usually in the
form of an ammonium salt.
A few bacteria can use gaseous nitrogen
or organic nitrogen (aminoacids or poly peptides) as a nitrogen source and reduce
it to ammonia.
5. Growth factors
Certain organic compounds such as aminoacids,
nucleotides, monosaccharides, lipids and coenzymes (essential nutrients) must be either
synthesized by the microorganism or provided as
nutrients in the environment.
Some microorganisms can synthesize by themselves all
the aminoacids and they are called prototrophs.
Auxotrophs have lost, by mutation, a biosynthetic ability
for one or more organic compunds.
Therefore these essential nutrients, called growth
factors, must be supplied in the growth medium.
5. Growth factors
Pathogenic bacteria are heterotrophs
dependent to several growth factors.
Some bacteria are so dependent to their natural
environment that they cannot be grown in vitro
(e.g., Treponema pallidum, Mycobacterium
leprae).
Auxotrophs have selective advantages: in the
presence of the necessary growth factor,
bacteria can multiply quicker than the parental
strain.
1. Temperature
The optimal temperature for growth of
some human pathogens is rather unique
and can be a simple way to select those
organisms.
For example, Campylobacter species
grow best at 42C, a temperature that is
excessive for the growth of most other
human pathogens.
1. Temperature
Mesophilic bacteria grow best at
temperature ranging from 20 C-40C.
Most human pathogens are mesophilic.
Thermophilic bacteria grow best at 50C60C.
Psychrophilic bacteria grow best at
temperatures ranging from 0C-10C.
3. Osmotic pressure
Bacteria are relatively tolerant of changes in
the osmotic pressure of their environment and
can grow in media with widely varying contents
of salt, sugar and other solutes.
This is partly a reflection of the mechanical
strength of their cell walls.
Halophilic species can grow at higher concentrations up to saturation; few halophilic species
are pathogens (e.g., Vibrio parahaemolyticus).
4. Oxygen
It is necessary to provide the correct atmosphere
for the growth of aerobes and anaerobes.
Aerobic and facultative anaerobic bacteria can
grow in the presence of oxygen.
For anaerobic bacteria, the presence of
oxygen is toxic.
The tolerance to oxygen is related to the ability of
the bacterium to detoxify superoxide and
hydrogen peroxide, produced by aerobic respiration.
BACTERIAL METABOLISM
The bacterial metabolism consists of catabolism
and anabolism.
Catabolism.
- Organic compounds (carbon source, energy
source) are degraded to simpler intermediates
that can be assimilated by the bacterium.
- This is done using hydrolytic enzymes
eliminated extracellular by the bacterial cell.
Anabolism
Simple structures are used for the synthesis of
compounds that are found in the bacterial
structure or function.
Anabolic reactions use energy.
The end-products of biosynthesis are very
similar for all bacteria.
However, there are wide differences in the ability
of cells to carry out the individual biosyntheses
of essential monomers and co-enzymes.
Anabolism
Some are capable of synthesizing all their
amino-acids, nucleotides, monosaccharides, coenzymes, and so on, from the building blocks
produced by catabolism.
Others almost completely lack such biosynthetic
powers and depend entirely on their nutrient
environment for the provision of such substances
in ready-made form.
With these two extremes there is a wide
spectrum of different biosynthetic abilities.
BACTERIAL GROWTH
The volume of the bacterial cell grows with
a cubic rate, while the surface grows with
a square rate.
The equilibrium between volume and
surface is brought by cell division (by
binary fission).
BACTERIAL GROWTH
Growth media are solutions that ensure
the nutrients necessary for the growth and
multiplication of bacteria.
Some bacteria grow well in simple media
(non-fastidious bacteria), whereas others
require complex media (fastidious
bacteria).
Bacterial cultivation
Inoculation of growth media is followed by
incubation; most bacterial species grow fast, in
18-24 hours at the optimum temperature.
Slowly growing bacteria need days or even
weeks (2 weeks minimum for Mycobacterium
tuberculosis).
Anaerobiosis is ensured by adding reducing
compounds (e.g., thyoglicolate) to the medium
or in anaerostates, in which the oxygen is
consumed by reducing substances.
Loop sterlization
Semisolid agar
Safety cabinet
Petri dishs
Pouring plates
Nutrient broth
Petri dish
Colonies types
Quantitative
A fix and known volume (V) of a certain dilution
(D) of the specimen is inoculated on the agar
plate.
The number of viable bacteria (X), in fact of the
colony forming units (CFU) in the specimen
results from:
X = N x D x 1/V
N = the number of colonies resulted on the
plate after incubation.
Quantitative
The number of total bacteria (dead and
alive) can be determined quicker
photoelectrically, by turbidimetric assays
or electronic counting.
Semiquantitative
After a four quadrant inoculation, growth
can be appreciated semi-quantitatively,
considering the number of colonies
resulted in each quadrant.
The method is easier, cheaper and
quicker than the quantitative
determination.