Nutrition and Culture of bacteria

By Fadilah Nor Laili Lutfia

Lecture of Microbiology
Nutrients: Chemical and Energy
Requirements
• Microbial cell: • Nutrition is required for growth.
• 70–80% of water • The minimum nutritional
• Macromolecules requirements are water, a source of
carbon, a source of nitrogen and some
• Proteins (55% of cell dry inorganic salts
weight) • Not all nutrients are required in the
• Nucleic acids same amounts:
• Lipids • Macronutrients, are required in
large amounts
• Polysaccharides
• Micronutrients, are required in
• Low molecular weight just trace amounts.
compounds
MACRONUTRIENT

Carbon and Nitrogen

• Carbon and nitrogen are important macronutrients
• Fatty acids
• Sugars
• Amino acids
• Nitrogen bases
• Source:
• Carbon: organic compound (heterotrophic), CO2 (autotrophic)
• Nitrogen: ammonia (NH3), nitrate (NO3), or nitrogen gas (N2)
MACRONUTRIENT

Other Macronutrients: P, S, K, Mg, Ca, Na

• Phosphorus : phosphate in nucleic acids and phospholipids
membrane
• Sulfur : amino acids cysteine and methionine and also in
several vitamins (thiamine, biotin, and lipoic acid)
• Potassium (K) : is required for the activity of several enzymes,
• Magnesium (Mg) : functions to stabilize ribosomes, membranes, and
nucleic acids and cofactor many enzymes.
• Calcium (Ca) : stabilize microbial cell walls
• Sodium (Na) : is required by some microorganisms, and its
typically of the habitat
Micronutrients and Growth Factors
Oxygen Requirements
• Oxygen: Organisms that use molecular oxygen as a final electron
acceptor in the process of respiration to produce ATP.
• Can classify microorganism based on their oxygen requirements:
1. Obligate Aerobes: Require oxygen to live
2. Facultative Anaerob: Can use oxygen, but can grow in its absence.
• Have complex set of enzymes. Examples: E. coli, Staphylococcus, yeasts, and many
intestinal bacteria.
3. Obligate Anaerobes: Cannot use oxygen and are harmed by the presence of
oxygen. Examples: Clostridium bacteria that cause tetanus and botulism.
4. Aerotolerant Anaerobes: Can’t use oxygen, but tolerate its presence.
5. Microaerophiles: Require oxygen, but at low concentrations. Example:
Campylobacter.
 Physical Requirements
1. Temperature
• Psychrophiles : Many bacteria
can grow fast at 4 °C
• Psychrotrop/Psychrotolerant
• Mesophiles : grow best at
temperatures between about
20°C and 40°C.
• Thermophiles : require
temperatures above 45°C.
• Hyperthermophiles: require
temperatures above 80°C.
2. pH

• Most bacteria prefer neutral pH (6.5-7.5)

• Acidophiles:
• “Acid loving”. Grow at very low pH (0.1 to 5.4). Ex: Lactobacillus produces lactic acid,
tolerates mild acidity.
• Neutrophiles:
• Grow at pH 5.4 to 8.5. Includes most human pathogens.
• Alkaliphiles:
• “Alkali loving”. Grow at alkaline or high pH (7 to 12 or higher). Ex: Vibrio cholerae optimal
pH 9, soil bacterium Agrobacterium grows at pH 12
3. Other Factors

• Halophiles:
• Require moderate to large salt concentrations.
• Ocean water contains 3.5% salt.
• Most bacteria in oceans.
• Extreme or Obligate Halophiles:
• Require very high salt concentrations (20 to 30%).
• Bacteria in Dead Sea, brine vats.
• Facultative Halophiles:
• Do not require high salt concentrations for growth, but tolerate 2% salt or more.
 Microbial Growth
In microbiology, growth is defined as an
increase in the number of cells.

This process is called

binary fission
(“binary” to express
the fact that two
cells have arisen
from one).

During cell division one cell becomes two (binary

fission). During the time that it takes for this to occur
(generation time or doubling time), both total cell
number and mass double
As an example:
Food contaminated with
Staphylococcus. If you ingest
100 cells then within a few
hours, the exponential growth
will produce more than 1.6
million cells, which will show
symptoms such as food
poisoning.
 Population Growth
• Lag Phase : When a microbial culture is inoculated into a
fresh medium, growth usually begins only after a period of Binary fission of
time called the lag phase. Staphylococcus aureus
• Exponential Phase : The exponential phase of growth each
cell divides to form two cells, each of which also divides to
form two more cells, and so on, for a brief or extended
period, depending on the available resources and other
factors
• Stationary Phase : In the stationary phase, there is no net
increase or decrease in cell number and thus the growth
rate of the population is zero.
• Death Phase : After a population reaches the stationary
phase, the cells may remain alive and continue to
metabolize, but they will eventually die. When this occurs,
the population enters the death phase of the growth cycle
 The Mathematics
of Exponential Growth
• The increase in cell number in an exponentially growing bacterial culture
approaches a geometric progression of the number 2. As one cell divides to
become two cells
N is the final cell number,
N0 is the initial cell number, and
n is the number of generations during
the period of exponential growth.

The generation time (g) of the exponentially growing

population is g = t/n
Limited Environment
Such unfavorable conditions:
• Present of toxic chemical
• Nutrient limitation

Endospora formation
(sporulation)
 MICROBIAL CULTURE
• Culture media are the nutrient solutions used to grow microorganisms in the
laboratory.
DEFINED MEDIA : Prepared by adding precise amounts of highly purified inorganic or
Classes of organic chemicals to distilled water.
Culture COMPLEX MEDIA: For culturing many microorganisms, complex medium is its imprecise
Media nutritional composition.

Enriched medium, often used for the culture of otherwise difficult-to-grow nutritionally
demanding (fastidious) microorganisms, with additional nutrients such as serum, blood

Selective medium, contains compounds that inhibit the growth of some microorganisms
but not others

Differential medium is one in which an indicator, typically a reactive dye, is added that
reveals whether a particular chemical reaction has occurred during growth.
Eosin methylene blue: E. coli is grown it will give a distinctive metallic green sheen by
lactose fermentation
CULTURE MEDIA PREPARATION
Solid and Liquid Culture Media
• Liquid culture media are sometimes solidified by the addition of a
gelling agent.
• Solid media immobilize cells, allowing them to grow and form visible,
isolated masses called colonies
• Solid media use for pure culture that population consisting only one
species
• Solid media are prepared in the same way as liquid media except that
before sterilization, agar, a gelling agent, is added to the medium,
typically at a concentration of 1–2%.
PURE CULTURE

Aseptic Technique

Procedure for removing

organisms from a broth
culture with inoculating loop
PURE CULTURE

Procedure for inoculating a

nutrient agar slant from an
agar plate
PURE CULTURE

STREAK PLATE METHOD

Procedure for viable counting using serial dilutions of
the sample and the pour-plate method

The sterile liquid used for making dilutions can simply be water,
but a solution of mineral salts or actual growth medium may
yield a higher recovery. The dilution factor is the reciprocal of
the dilution.
 A viable cell is one that is able to divide and form offspring, and in most cell-
counting situations, these are the cells we are most interested in. For these
purposes, we can use a viable counting method.

Two methods for the viable count

In the pour-plate method, colonies form within the agar as well as on the agar surface. On the far right are photos of colonies of
Escherichia coli formed from cells plated by the spread-plate method (top) or the pour-plate method (bottom).
DIRECT MICROSCOPIC COUNT
Turbidimetric Methods/
Indirect Methods
Method of Culture
1. A batch culture is a closed system.
In the early stages of exponential growth in batch cultures, conditions may
remain relatively constant. But in later stages, when cell numbers become
quite large, the chemical and physical composition of the culture medium
changes dramatically.

2. A continuous culture is an open system.

The continuous culture vessel maintains a constant volume to which fresh
medium is added at a constant rate while an equal volume of spent culture
medium (containing cells) is removed at the same rate. Once such a system is
in equilibrium, the chemostat volume, cell number, and nutrient status
remain constant, and the system is said to be in steady state.
Schematic for a continuous culture device (chemostat)
The population density is controlled by the concentration of
limiting nutrient in the reservoir, and the growth rate is
controlled by the flow rate.
THANKS
TUGAS