LECTURE 4. MICROBIAL GROWTH
1. Microbial survival strategies
2. Microbial growth
3. Effects of the environment
4. Microbial cultures
5. Growth measurements
6. Antimicrobial agents
7. Antibiotics
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
1. MICROBIAL SURVIVAL STRATEGIES
PROKARYOTES: MINIATURIZATION
EUKARYOTES: COMPLEXITY
Prokaryotes – r strategists: “the advantages of being small”
 Surface/Volumen
Metabolic rates (osmotrophs)
Rapid growth, short generation times
Population-level adaptation
Eukaryotes –K strategists: “the advantages of being complex”
Phagocytosis
Complex genomes
Rapid movement
Independence from the environment
Complex sensor and motor systems
Individual-level adaptation
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
2. MICROBIAL GROWTH
GROWTH (IN MICROBIOLOGY)=INCREASE IN CELL NUMBER
2.1. CELL DIVISION AND PARTITION OF CELL COMPONENTS
DNA replication and cell
elongation
Septum formation
Completion of septum with
formation of distinct walls
Cell separation
Binary fission vs other
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
2. MICROBIAL GROWTH
2.1. CELL DIVISION AND PARTITION OF CELL COMPONENTS
Partition of cell components: random (except DNA)
PROTEINS Fts
DNA SEGREGATION:
DNA binds to the cytoplasmic membrane
 Bidirectional replication (in one fork)
 Several simultaneous forks
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
3. EFFECTS OF THE ENVIRONMENT
3.1. NUTRIENT CONCENTRATION
At a very low nutrient concentration, permeases
cannot keep high levels of nutrients inside the cell
and growth rate decreases.
However, high nutrient concentrations can be toxic for
many microorganisms
3.2. PREASURE
Al sea level = 1 atm
In the oceans up to 600 -1.100 atm
• No barotolerant
• Barotolerant
• Barophile (or piezophile)
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
3. EFFECTS OF THE ENVIRONMENT
3.3. TEMPERATURE
Enzymatic reactions
occurring at maximum
possible rate
Enzymatic reactions
occrring at increasingly
rapid rates
Membrane gelling;
transport processes
so slow that growth
cannot occur
Protein denaturation;
collapse of the cytoplasmic
membrane;thermal lysis
• Psychrophile ( 0 - 20ºC)
• Mesophile (10 - 50ºC)
• Termophile (50 - 70ºC)
• Hyperthermophile (80 121ºC*)
Adaptations to high temperatures
Adaptations to low temperatures
Thermoresistent proteins (enzymes)
Proteins (enzymes) that function optimally in
the cold
Stable membranes ( saturated fatty acids)
Archaea, special membranes (lipid
monolayers)
Modified active transport processes
Fluidity of membranes ( unsaturated fatty
acids)
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
3. EFFECTS OF THE ENVIRONMENT
3.4. OXYGEN CONCENTRATION
TOXIC OXYGEN SPECIES:
Singlet oxygen (1O2)
Superoxide (O2-)
Hydrogen peroxide (H2O2)
Hydroxyl radical (OH-)
Enzymes that destroy them
1
2
3
4
5
Aerobic (1)
Microaerophilic (aerobic) (4)
Facultative (3)
Aerotolerant anaerobe(5)
(Strict) anaerobic (2)
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
3. EFFECTS OF THE ENVIRONMENT
3.5. pH
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
3. EFFECTS OF THE ENVIRONMENT
3.6. OSMOLARITY
Adaptations to high salt
Counterbalance of external osmotic preasure
by accumlation of:
-Inorganic ions (K+) . Acidic proteins! Archaea
and some extremely halophilic Bacteria.
-Compatible organic solutes (either imported or
synthesized): glycine betaine, proline, glycerol,
etc.
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
3. EFFECTS OF THE ENVIRONMENT
3.7. RADIATIONS
Photodynamic effect: light-mediated generation of singlet oxygen ( 1O2 )
Carotenoids: photoprotectant pigments
transform 1O2 into non toxic species
Radiotolerant microorganisms:
Bacterial endospores
Deinococcus radiodurans
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
3. EFFECTS OF THE ENVIRONMENT
3.8. EXTREMOPHILISM AND EXTREMOPHILES
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
4. MICROBIAL CULTURES
CULTURE: a system used to allow the multiplication of a microbial population
and reach a high microbial density
Culture components:
Nutrients (medium)
Inoculum
(absence of contamination)
(According to the metabolic categories):
C source
E source
Macronutrients (N, O, P, S, salts, vitamins, etc.)
Micronutrients (normally, present as salt
contaminants)
H2O
pH (buffers)
WARNING! Auxotrophs vs prototrophs
Types of culture media:
Liquid / solid
Defined, synthetic
Complex
Selective
“Test”…
Culture types:
Pure (or axenic)
Mixed
Preparation
Sterilization
Inoculation
Incubation
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
4. MICROBIAL CULTURES
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
4. MICROBIAL CULTURES
4.1. BATCH (DISCONTINUOUS) CULTURE
Closed system (only energy, and sometimes gases, are interchanged with the
external environment; no cells or disolved products).
Growth curve with 4 phases.
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
4. MICROBIAL CULTURES
4.1. BATCH CULTURE
Exponential
growth
N = N02n
N= Number of cells after n generations
N0 = Number of cells at the beginning
tg = Generation time
(time needed to double cell number)
= specific growth rate (time units -1)
(number of generations per time unit)
tg = ln 2 /  (hours)
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
4. MICROBIAL CULTURES
4.1. BATCH CULTURE
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
4. MICROBIAL CULTURES
4.1. BATCH CUTURE
Escherichia coli Tg= 20 min
After 48 hours
4000 X Earth weight
µ depends on
nutrient
concentration
LIMITING SUBSTRATE
Net growth: final biomass – initial biomass (inoculum)
Yield: unit of biomass produced per unit of nutrient
consumed
Y = (X – Xo) / S
Y = yield
X, Xo = cells/ ml
S = nutrient concentration at to
µ = µ max
S
Ks + S
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
4. MICROBIAL CULTURES
4.2. CONTINUOUS CULTURE (“steady state”)
Cultures can be kept for long periods of time. Medium is added and culture
removed, keepin V constant
V entrance constant, [nutrient] constant, [cell] constant.
V changes, µ changes and a new [cell] is reached
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
4. MICROBIAL CULTURES
4.2. CONTINUOUS CULTURE (“steady state”)
Biomass (X) constant in time. dX/dt = 0
Vproduction (cells produced) = Vlosses (cells removed)
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
4. MICROBIAL CULTURES
4.3. CULTURES ON SOLID MEDIA
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
5. GROWTH MEASUREMENTS
Only balanced growth (ordered increase of all cell components) can be
measured properly…
5.1. BIOMASS
Dry weight
Absorbance (cell density)*
5.2. CELL COMPONENTS
Nucleic acids, proteins, enzymatic activities
5.3. CELL NUMBRES
Total cells
Counting chamber
Flow cytometer
Viable cells
(culturable)***
***VBNC: Viable but not culturable
Plate counts
Most probable number (MPN)
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
5. GROWTH MEASUREMENTS
5.3. CELL NUMBERS
Description of natural
microbial communities
How many microbes are present in a natural sample?
“Who” are they?
What do they do? (niche)
How do they relate to each other and to other organisms?
(competition, antagonisms, symbioses, etc.)
Plate count (“viables”)
SAMPLE
Direct counts
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
Problems encountered when counting “viables” (i.e. when culturing)
Are they dead?
Are they viable but not culturable (VBNC)*?
They do not grow on standard culture media
*Important in public health
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
ANTIMICROBIAL AGENTS: either (i) limit or inhibit microbial growth or (ii)
destroy microorganisms
IMPORTANT CONCEPTS
•
Sterilization: a process that destroys all living organisms and their viruses from an
object or habitat.
•
Disinfection: partial elimination or inhibition of microbes, normally pathogens
Disinfectant: (chemical) agents used to disinfect; used on inanimate objects.
•
Antisepsis: prevention of sepsis or infection (antiseptic agents are used over
tissues to prevent infections, normally less toxic than disinfectant agents).
•
Germicide: destroy germs (pathogens) and non-pathogens, but not necessarily
spores (bactericide, algaecide, fungicide, virocide...)
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
ANTIMICROBIAL AGENTS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
6.1. EFFECTS OF ANTIMICROBIAL AGENTS
BACTERIOSTATIC
BACTERICIDE
BACTERIOLYTIC
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
6.2. FACTORS THAT AFFECT THE EFFICIENCY OF ANTIMICROBIAL AGENTS
•
Population size: the same fraction of the microbial population is destroyed in each
time interval; a larger population needs more time to be completely eliminated than a
smaller one.
•
Population composition: different microbes have different sensitivity to antimicrobial
agents
Concentration and performance of the antimicrobial agent
Exposure time
Temperature
Local environment: pH, organic matter, biofilms, etc.
•
•
•
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L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS
MOIST HEAT
Boiling in water for 10
minutes destroys
vegetative cells and
eukaryotic spores but
NOT bacterial
endospores
Autoclave: temperatures higher than
100oC (pressure) with water saturated
steam. Time: 10-15 minutes. Depends on
the sample volume.
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS
PASTEURIZATION
NO STERILIZATION
Food treatment (milk...)
Old method: 63oC for 30 minutes.
Fast pasteurization(HTST: high-temperature short-term): 72oC for 15 seconds.
Sterilization at ultrahigh temperature (UHT: ultra-high temperature): 140-150oC for 1-3
seconds.
DRY HEAT
Oven at 160-170 oC from 2 to 3 hours
Not suitable for thermosensitive materials
Used for glass, oil and other materials Suele utilizarse para material de vidrio, aceite y otros
materiales
Clostridium botulinum endospores
Moist heat: 5 min at 121 oC
Dry heat: 2 hours at 160oC
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS
LOW TEMPERATURES
Inhibit growth
Used to preserve (not to sterilize or disinfect)
FILTRATION
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
6.3. STERILIZATION AND DISINFECTION BY PHYSICAL AGENTS
RADIATION
UV (ceiling, biological safety hoods)
Ionizing radiation: very good sterilizing agent.
Pharmaceutical companies
Disposable clinical materials
Meat and other foods (spices)
Comercial radiation of
spices and
seasonings, world data
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
6.3. STERILIZATION AND DISINFECTION BY CHEMICAL AGENTS
Germicides
Disinfectant and antispetic
Antibiotics
•
•
•
•
•
•
•
•
•
Phenols
Alcohols
Halogenated compounds
Heavy metals
Aldehydes
Hydrogen peroxide
Surfactant agents
Ethiylene oxides
ANTIBIOTICS
NO SELECTIVE TOXICITY
SELECTIVE TOXICITY
STERILIZING AGENTS
Ethylene oxide
Formaldehyde
Glutaraldehyde
H2O2 30%
DISINFECTANT AGENTS
Alcohols
Chlorinated compounds
Phenolic compounds
H2O2 6%
ANTISEPTIC AGENTS
Mercury-containing compounds
Iodine
H2O2 3%
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
6.3. STERILIZATION AND DISINFECTION BY CHEMICAL AGENTS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
6. CONTROL OF MICROBIAL GROWTH
6.5. MEASURING ANTIMICROBIAL ACTIVITY
MINIMUM INHIBITORY
CONCENTRATION
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
A chemical substance produced by a microorganism (fungi or bacteria) that kills or inhibits the growth
of another microorganism.
Normally, they have selective toxicity*: the ability of a compound to inhibit or kill pathogenic
microorganisms without adversely affecting the host. Thus, they can be used as chemotherapeutical
agents.
Some antibiotics are semi-synthetic.
“*The magic bullet”
BACTERIOSTATIC
BACTERICIDE
BACTERIOLYTIC
MAIN ANTIBIOTIC TARGETS
Cell wall synthesis
Protein synthesis
Cell membrane integrity
Nucleic acids synthesis
Essential cofactors synthesis
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
ANTIBIOTIC MECHANISMS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
7.1. CELL WALL (PETIDOGLYCAN) SYNTHESIS INHIBITORS
PENICILLINS (b-LACTAMIC)
b-lactamic ring (degraded by b-lactamases or
penicillinases)
Penicilina G
Synthesized by the fungus Penicillium (Fleming,
1928)
Bacteriolytic (destroy growing cells)
They inhibit transpeptidation
Bacteria can be resistant to penicillins if they
synthesize penicillinases (b-lactamases)
They can be combined with clavulanic acid
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
7.1. CELL WALL (PETIDOGLYCAN) SYNTHESIS INHIBITORS
PENICILLINS (b-LACTAMIC)
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
7.1. CELL WALL (PETIDOGLYCAN) SYNTHESIS INHIBITORS
CEPHALOSPORINS
Cephalosporium
acreminium
They inhibit transpeptidation
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
7.2. PROTEIN SYNTHESIS INHIBITORS
AMINOGLUCOSIDES
The effect cannot be reverted
Examples: streptomycin, kanamycin, etc.
TETRACICLINES
The effect can be reverted
MACROLIDES
CHLORAMPHENICOL
Erythromycin
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
7.3. OTHER MECHANISMS
Changes in the properties of the cell membrane:
- Polymixin B
Interference with nucleic acids synthesis:
- Rifampicin (inhibits RNA polymerase)
- Quinolones (inhibit DNA topoisomerases)
Inhibit essential cofactors synthesis:
- Sulfamides (inhibit folic acid synthesis)
7.4. BACTERIOCINS
Agents produced by certain bacteria (or archaea) that inhibit or kill closely
related species.
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTIC
7.5. ANTIFUNGAL AGENTS
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
7.6. ANTIVIRAL
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
7.7. ANTIBIOTIC RESISTANCE
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014
7. ANTIBIOTICS
7.7. ANTIBIOTIC RESISTANCE
RESISTANCE MECHANISMS
1. The antibiotic cannot reach
its target
2. The antibiotic is degraded or
modified
3. The antibiotic target is
modified
Antibiotic resitance can be
chromosomic (mutation) or
plasmidic (transferable)
Plasmids R
Can we stop
antibiotic
resistance?
L. 4: Microbial Growth. Microbiology. 2nd Biology ARA 2013-2014