Nutrition and bacteria

cultivation
Culture media

Purposes of bacterial cultivation:

Medical purposes:

Identification and;
susceptibility testing to antimicrobial drugs;

For industry, to obtain:

biosynthesis products antibiotics, amino-acids;

bioconversion products steroid hormones;
fermentation products alcohols, organic acids;
vaccines.

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.

** The electron acceptor

The oxygen is used in aerobic respiration.
The respiratory chain of electron transport
associated to the cytoplasmic membrane is
implicated.
Organic compounds (the carbon source or its
metabolites) are used in fermentative processes.
Fermentation results in a mixture of final
products, some more reduced, some more
oxidized. It is less efficient than aerobic
respiration for obtaining the energy.

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.

4. Other inorganic salts

Microorganisms require a supply of inorganic
salt for growth, particularly the anions phosphate
and sulfate, and the cations sodium, potassium,
magnesium, iron, and calcium.
Some ions such as cobalt, zinc, manganese,
copper are needed in trace amounts and are
supplied by the tap water or impurities from the
culture medium or glassware.

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.

PHYSICAL CONDITIONS REQUIRED

FOR GROWTH
1. Temperature
2. Hydrogen ion concentration (pH)
3. Osmotic pressure
4. Oxygen

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.

2. Hydrogen ion concentration (pH)

The majority of commensals and pathogenic
bacteria grow best at a neutral or very slightly
alkaline reaction (pH 7.2-7.6).
Some bacteria grow in the presence of a
considerable degree of acidity and are termed
acidophilic, e.g. Lactobacillus species.
Some species are very sensitive to acid, but
tolerant of alkali, e.g. Vibrio cholerae (pH - 9).

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).

Classification of growth media:

origin of nutrients: empirical and synthetic;
consistency: liquid, semi-solid, solid.
liquid media (broth) are preferred for monobacterial
specimens (blood, CSF);
growth on solid media (agar) is preferred when bacterial
colonies are to be isolated or further characterized
(multibacterial specimens);
composition:
simple media (e.g., nutrient agar)
enriched media (e.g., blood agar)
special media: isolation media
differential media (BBTLA)
selective media (DCA)
enrichment media (selenite broth)
identification media

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.

Different culture media

Liquid culture media

Culture media kit (liquid, solid,

semisolid, identification media)

Microbiological needle and loop

Loop sterlization

Pass the mouths of the tubes

through the flame.

Remove a sample from a broth

culture by using a sterile wire loop

Use either loop or needle to remove a sample from an agar slant.

Touch the colony to be subcultured with the wire but do not break the
surface of the agar. Use a needle to transfer to a deep tube
Reflame the mouths of the tubes and replace the caps.

Reflame the loop or needle before

putting it down.

Notes on Transferring Samples

Transferring to broth Put the loop in the
broth and then swirl it.
Agar slant or plate When inoculating an
agar slant or plate, draw the loop very
lightly over the surface while being careful
not to break the surface. A straight or zigzag motion can be used.

Deep Agar Tube Push needle directly into

the agar to the bottom of the tube, then
remove it.

Semisolid agar

Safety cabinet

Petri dishs

Pouring plates

Nutrient broth

Petri dish

Petri dish - schematic incoculation

to obtaine isolated colonies

Appreciation of bacterial growth

Qualitative. Liquid media become turbid;
on the surface of solid media appear
colonies.

Appreciation of bacterial growth

Bacteria with a negatively charged and
hydrophilic surface (due to the presence
of antigen O) form S (smooth) cultures.
Bacteria with reduced electronegativity
and hydrophilia of the surface form R
(rough) cultures.
Colonies are differentiated by: size, color,
contour, surface, opacity, relief,
consistency, haemolysis.

Colonies types

Klebsiella pneumonie - mucoid

colonies (growth in special culture
media)

Proteus - invasive growth in

nonselective culture media

Yersinia pestis - fried eggs

colonies

Lactose positive red colonies (Escherichia

coli) and lactose negative us culture media
(Salmonella) colonies in differential
selective culture media

E coli O157H7 chromogenic

media (purple colonies)

E. coli blue colonies in other

chromogenic culture media

Identification tests (TSI triple

sugars iron)

Synthetic culture media

(ex Simmons citrate is used as C
source)

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.

Semiquantitative the growth until

last square = 106 CFU inoculated
sample

BACTERIAL GROWTH CYCLE

Bacterial cultures are discontinous, when
realised in a limited volume of medium or
continous, when there is a continous supply of
fresh nutrients into the culture vessel and a
continous removal of grown bacteria by means
of a constant-level device (in a chemostat).
During typical bacterial growth, bacterial cells
divide by binary fission and their mass and
number increase in an exponential manner.
Bacterial growth in culture can be separated into
at least four distinct phases relative to the
introduction of the bacteria to the medium.

BACTERIAL GROWTH CYCLE

Lag phase.
Logarithmic (exponential) phase.
Stationary phase
Decline or death phase

Lag phase (A)

This is a period of intense physiologic
adjustement involving the introduction of
new enzymes and the synthesis and
assembly of ribosomes.
There is no appreciable multiplication of
cells.

Lag phase (A)

Logarithmic (exponential) phase

(B)
This phase is characterized by maximal rates of cell
division.
The generation time (i.e., the time required for doubling
the number of bacteria) during the logarithmic phase is
constant for a given bacterial species grown under the
same set of conditions, but varies among species (e.g.,
20 minutes for Escherichia coli, 27 hours for
Mycobacterium tuberculosis).
Bacteria are very susceptible to antimicrobial
agents.
In this stage will be useful for testing the
susceptibility to antibiotics!

Logarithmic (exponential) phase.

Stationary phase (C)

During this period, the availability of
essential nutrients becomes a limiting
factor, and there is a balance between cell
growth and division and cell death.
Bacteria have typical morphology for
the species; the susceptibility to
antimicrobial drugs decreases.
In this stage will be useful for smear
examination!

Stationary phase (C)

Decline or death phase.

During this phase, bacterial cells undergo
lysis, which reduces the number of viable
cells.

Decline or death phase.